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Perkin, L. C., B. Oppert, S. Duke, and C. P.-C. Suh. "Assessment of DNA Integrity From Trap-Captured Boll Weevil (Coleoptera: Curculionidae) for Use in a New PCR-Based Diagnostic Tool." Journal of Economic Entomology 114, no. 3 (2021): 1321–28. http://dx.doi.org/10.1093/jee/toab073.
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Abstract The boll weevil, Anthonomus grandis grandis Boheman (Coleoptera: Curculionidae), is a major pest of commercial cotton (Gossypium hirsutum) in the southern United States and throughout Central and South America. Efforts are underway to develop a PCR-based diagnostic tool that can be used to rapidly and accurately differentiate boll weevils from other weevil species that are commonly captured in pheromone traps. However, the quantity and integrity of weevil DNA must be sufficient for a successful PCR assay. Currently, active eradication programs service traps weekly, but post-eradication programs service traps at 2- or 3-wk intervals. Consequently, captured weevils may be dead, dismembered, and exposed to environmental conditions for prolonged periods which may adversely affect the quantity and quality of weevil DNA. We documented DNA quantity and integrity in boll weevils and weevil body parts aged in traps over a 3-wk period under field conditions. The quantity of DNA extracted from whole weevils, heads, abdomens, and legs generally remained sufficient (> 1 ng/μl) for successful PCR amplification throughout the 21-d period. The integrity (fragment length) of extracted DNA declined over time but generally was sufficient (> 700 bp) for successful amplification. PCR amplification of three marker genes validated that the quality and integrity of DNA extracted from dead weevils and individual weevil body parts aged in traps up to 21 d remained at sufficient levels for the PCR-based assay. However, our data also suggested that rain events may accelerate degradation of weevil DNA.
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Du, Xiongwei, Hongjie An, Bo Jin, Liangyu Meng, and Qingdai Liu. "Carbon Nanotubes Altering Specificity of Repeated PCR and DNA Integrity Properties." Journal of Nanoscience and Nanotechnology 14, no. 7 (2014): 5547–51. http://dx.doi.org/10.1166/jnn.2014.8874.
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Bergerová, E., Z. Godálová, and P. Siekel. "Combined effects of temperature, pressure and low pH on the amplification of DNA of plant derived foods." Czech Journal of Food Sciences 29, No. 4 (2011): 337–45. http://dx.doi.org/10.17221/217/2010-cjfs.
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The effect of food processing on the DNA integrity was studied by means of PCR amplification of soybean, transgenic MON 810 and non-transgenic maize, bean, and pea. The degree of DNA degradation was checked by PCR and visualised by agarose gel electrophoresis. The conditions of technological treatment such as temperature, pH, pressure, and their combination may negatively influence the integrity of DNA in processed foods and hence PCR detection of food components. The DNA over 300 bp was amplifiable when mild processing parameters up to 100°C were performed at approximately neutral or low acidic pH. The autoclaving (12°C; 0.1 MPa) significantly reduced the size of amplifiable DNA in the time dependant manner and that was intensified by acidic pH. The maximum amplicons length achieved for highly processed matrices was 300 bp. The major impact on the DNA integrity was exerted by the combination of pressure, temperature, and low pH.
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Frasquilho, Sonia G., Ignacio Sanchez, Changyoung Yoo, Laurent Antunes, Camille Bellora, and William Mathieson. "Do Tissues Fixed in a Non-crosslinking Fixative Require a Dedicated Formalin-free Processor?" Journal of Histochemistry & Cytochemistry 69, no. 6 (2021): 389–405. http://dx.doi.org/10.1369/00221554211017859.
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We evaluate the consequences of processing alcohol-fixed tissue in a processor previously used for formalin-fixed tissue. Biospecimens fixed in PAXgene Tissue Fixative were cut into three pieces then processed in a flushed tissue processor previously used for formalin-fixed, paraffin-embedded (FFPE) blocks (neutral buffered formalin [NBF]+ve), a formalin-free system (NBF−ve), or left unprocessed. Histomorphology and immunohistochemistry were compared using hematoxylin/eosin staining and antibodies for MLH-1, Ki-67, and CK-7. Nucleic acid was extracted using the PAXgene Tissue RNA/DNA kits and an FFPE RNA extraction kit. RNA integrity was assessed using RNA integrity number (RIN), reverse transcription polymerase chain reaction (RT-PCR) (four amplicons), and quantitative RT-PCR (three genes). For DNA, multiplex PCR, quantitative PCR, DNA integrity number, and gel electrophoresis were used. Compared with NBF−ve, RNA from NBF+ve blocks had 88% lower yield and poorer purity; average RIN reduced from 5.0 to 3.8, amplicon length was 408 base pairs shorter, and Cq numbers were 1.9–2.4 higher. Using the FFPE extraction kit rescued yield and purity, but RIN further declined by 1.1 units. Differences between NBF+ve and NBF−ve in respect of DNA, histomorphology, and immunohistochemistry were either non-existent or small in magnitude. Formalin contamination of a tissue processor and its reagents therefore critically reduce RNA yield and integrity. We discuss the available options users can adopt to ameliorate this problem:
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Raja, Siva, Jesus Ching, Liqiang Xi, et al. "Technology for Automated, Rapid, and Quantitative PCR or Reverse Transcription-PCR Clinical Testing." Clinical Chemistry 51, no. 5 (2005): 882–90. http://dx.doi.org/10.1373/clinchem.2004.046474.
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Abstract Background: PCR-based assays can improve clinical care, but they remain technically demanding and labor-intensive. We describe a new instrument, the GeneXpert®, that performs automated nucleic acid isolation, reverse transcription, and fluorescence-based quantitative PCR in ∼35 min. Methods: Yield and integrity of RNA isolated on the GeneXpert were compared with Qiagen-based extraction for parallel samples (5-μm frozen tissue sections). The reproducibility of automated RNA isolation, reverse transcription, and quantitative PCR was determined by replicate (n = 10) analysis of 10 tissues, using duplex (target and endogenous control) reverse transcription-PCR reactions for two gene combinations. The GeneXpert was then used to perform rapid analysis of lymph nodes from melanoma, breast cancer, and lung cancer patients and analysis of melanoma metastatic to the lung, primary lung adenocarcinoma, and healthy lung tissue. Results: On the GeneXpert, RNA was recovered in slightly over 6 min, and the yield was ∼70% of that from parallel Qiagen reactions. The RNA integrity was comparable to that of Qiagen-isolated RNA as determined by gel electrophoresis. For the melanoma samples, the 95% prediction interval for the ΔCt for a new measurement was ±1.54 cycles, and for breast cancer samples, the interval for a newly observed ΔCt was ±1.40 cycles. GeneXpert assays successfully detected the presence of metastatic melanoma, breast cancer, and lung cancer in lymph nodes and also differentiated among metastatic melanoma, lung adenocarcinoma, and healthy lung. Conclusions: RNA yield and integrity on the GeneXpert are comparable to benchtop methods. Reproducibility of the GeneXpert data is similar to that seen with manual methods in our hands but may need improvement for some applications. The GeneXpert can perform RNA isolation, reverse transcription, and quantitative PCR in ∼35 min and could therefore be used for intraoperative testing when applicable.
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Fleige, Simone, and Michael W. Pfaffl. "RNA integrity and the effect on the real-time qRT-PCR performance." Molecular Aspects of Medicine 27, no. 2-3 (2006): 126–39. http://dx.doi.org/10.1016/j.mam.2005.12.003.
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Gorzelak-Pabis, Paulina, Emilia Luczak, Katarzyna Wojdan, et al. "Endothelial integrity may be regulated by a specific antigen via an IgE-mediated mechanism." Postępy Higieny i Medycyny Doświadczalnej 71, no. 1 (2017): 0. http://dx.doi.org/10.5604/01.3001.0010.3800.
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Background: Human vascular endothelial function and integrity may be regulated by many non-specific factors. However, the potential influence of specific antigens via an IgE-mediated mechanism remains unknown. The aim of the study was to determine the expression of the IgE receptors FcεRI and FcεRII in the human vascular endothelium and to assess their relevance in the IgE-mediated regulation of endothelial integrity.Material/Methods: FcεRI and FcεRII expression in human umbilical vein endothelial cells (HUVEC) was genetically assessed by PCR with respective primers and sequencing. HUVEC were cultured with IL-4, and changes in FcεRI and FcεRII mRNA expression were analyzed by real-time PCR. Changes in the integrity of endothelium pre-treated with anti-BSA-DNP IgE following exposure to the specific BSA-DNP antigen was assessed using the Real-time Cell Electric Impedance Sensing system (RTCA-DP).Results: PCR and sequencing revealed the expression of FcεRI and FcεRII receptors in the human vascular endothelium. IL-4 caused respective 2- and 3-fold increases in FcεRI and FcεRII mRNA expression. Exposure of endothelium pre-treated with anti-BSA-DNP IgE to specific BSA-DNP antigen led to a 20% increase of endothelial integrity (p<0.05) after 24 hours, but only in cells pre-incubated with IL-4.Conclusions: The presence of FcεRI and FcεRII may allow the human vascular endothelium to respond to a specific antigen by increasing its integrity via an IgE-mediated mechanism.
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Oakey, H. Jane. "A Universal Test to Determine the Integrity of RNA, and its Suitability for Reverse Transcription, in Animal Tissue Laboratory Specimens." Journal of Veterinary Diagnostic Investigation 19, no. 5 (2007): 459–64. http://dx.doi.org/10.1177/104063870701900501.
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Degradation of RNA in diagnostic specimens can cause false-negative test results and potential misdiagnosis when tests rely on the detection of specific RNA sequence. Current molecular methods of checking RNA integrity tend to be host species or group specific, necessitating libraries of primers and reaction conditions. The objective here was to develop a universal (multi-species) quality assurance tool for determining the integrity of RNA in animal tissues submitted to a laboratory for analyses. Ribosomal RNA (16S rRNA) transcribed from the mitochondrial 16S rDNA was used as template material for reverse transcription to cDNA and was amplified using polymerase chain reaction (PCR). As mitochondrial DNA has a high level of conservation, the primers used were shown to reverse transcribe and amplify RNA from every animal species tested. Deliberate degradation of rRNA template through temperature abuse of samples resulted in no reverse transcription and amplification. Samples spiked with viruses showed that single-stranded viral RNA and rRNA in the same sample degraded at similar rates, hence reverse transcription and PCR amplification of 16S rRNA could be used as a test of sample integrity and suitability for analysis that required the sample's RNA, including viral RNA. This test will be an invaluable quality assurance tool for determination of RNA integrity from tissue samples, thus avoiding erroneous test results that might occur if degraded target RNA is used unknowingly as template material for reverse transcription and subsequent PCR amplification.
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Cédile, Oriane, Sólja Remisdóttir Veyhe, Marcus Høy Hansen, Kjell Titlestad, and Charlotte Guldborg Nyvold. "Investigation of circulating DNA integrity after blood collection." BioTechniques 71, no. 5 (2021): 550–55. http://dx.doi.org/10.2144/btn-2020-0167.
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Method summary Concentrations of circulating DNA in blood plasma were compared using NanoDrop, Qubit, quantitative PCR and Bioanalyzer, and DNA integrity was evaluated with the Bioanalyzer according to the time of plasma preparation.
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Didelot, Audrey, Steve K. Kotsopoulos, Audrey Lupo, et al. "Multiplex Picoliter-Droplet Digital PCR for Quantitative Assessment of DNA Integrity in Clinical Samples." Clinical Chemistry 59, no. 5 (2013): 815–23. http://dx.doi.org/10.1373/clinchem.2012.193409.
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BACKGROUND Assessment of DNA integrity and quantity remains a bottleneck for high-throughput molecular genotyping technologies, including next-generation sequencing. In particular, DNA extracted from paraffin-embedded tissues, a major potential source of tumor DNA, varies widely in quality, leading to unpredictable sequencing data. We describe a picoliter droplet–based digital PCR method that enables simultaneous detection of DNA integrity and the quantity of amplifiable DNA. METHODS Using a multiplex assay, we detected 4 different target lengths (78, 159, 197, and 550 bp). Assays were validated with human genomic DNA fragmented to sizes of 170 bp to 3000 bp. The technique was validated with DNA quantities as low as 1 ng. We evaluated 12 DNA samples extracted from paraffin-embedded lung adenocarcinoma tissues. RESULTS One sample contained no amplifiable DNA. The fractions of amplifiable DNA for the 11 other samples were between 0.05% and 10.1% for 78-bp fragments and ≤1% for longer fragments. Four samples were chosen for enrichment and next-generation sequencing. The quality of the sequencing data was in agreement with the results of the DNA-integrity test. Specifically, DNA with low integrity yielded sequencing results with lower levels of coverage and uniformity and had higher levels of false-positive variants. CONCLUSIONS The development of DNA-quality assays will enable researchers to downselect samples or process more DNA to achieve reliable genome sequencing with the highest possible efficiency of cost and effort, as well as minimize the waste of precious samples.
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Du Cheyne, Charis, Yao Chen, Jurgen De Craene, Olivier Thas, and Ward De Spiegelaere. "Development of a 3’:5’ digital PCR assay to determine horse mRNA integrity." Analytical Biochemistry 626 (August 2021): 114217. http://dx.doi.org/10.1016/j.ab.2021.114217.
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Tereshko, Lauren, Xiaohui Zhao, Jake Gagnon, et al. "A novel method for quantitation of AAV genome integrity using duplex digital PCR." PLOS ONE 18, no. 12 (2023): e0293277. http://dx.doi.org/10.1371/journal.pone.0293277.
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Recombinant adeno-associated virus (rAAV) vectors have become a reliable strategy for delivering gene therapies. As rAAV capsid content is known to be heterogeneous, methods for rAAV characterization are critical for assessing the efficacy and safety of drug products. Multiplex digital PCR (dPCR) has emerged as a popular molecular approach for characterizing capsid content due to its high level of throughput, accuracy, and replicability. Despite growing popularity, tools to accurately analyze multiplexed data are scarce. Here, we introduce a novel statistical model to estimate genome integrity from duplex dPCR assays. This work demonstrates that use of a Poisson-multinomial mixture distribution significantly improves the accuracy and quantifiable range of duplex dPCR assays over currently available models.
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Prantner, Andrew, and Dianna Maar. "Genome concentration, characterization, and integrity analysis of recombinant adeno-associated viral vectors using droplet digital PCR." PLOS ONE 18, no. 1 (2023): e0280242. http://dx.doi.org/10.1371/journal.pone.0280242.
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Precise, reproducible characterization of AAV is critical for comparing preclinical results between laboratories and determining a safe and effective clinical dose for gene therapy applications. In this study, we systematically evaluated numerous parameters to produce a simple and robust ddPCR protocol for AAV characterization. The protocol uses a low ionic strength buffer containing Pluronic-F68 and polyadenylic acid to dilute the AAV into the ddPCR concentration range and a 10-minute thermal capsid lysis prior to assembling ddPCR reactions containing MspI. A critical finding is that the buffer composition affected the ITR concentration of AAV but not the ITR concentration of a double stranded plasmid, which has implications when using a theoretical, stoichiometric conversion factor to obtain the titer based on the ITR concentration. Using this protocol, a more comprehensive analysis of an AAV vector formulation was demonstrated with multiple ddPCR assays distributed throughout the AAV vector genome. These assays amplify the ITR, regulatory elements, and eGFP transgene to provide a more confident estimate of the vector genome concentration and a high-resolution characterization of the vector genome identity. Additionally, we compared two methods of genome integrity analysis for three control sample types at eight different concentrations for each sample. The genome integrity was independent of sample concentration and the expected values were obtained when integrity was determined based on the excess number of positive droplets relative to the number of double positive droplets expected by chance co-encapsulation of two DNA targets. The genome integrity was highly variable and produced unexpected values when the double positive droplet percentage was used to calculate the genome integrity. A protocol using a one-minute thermal capsid lysis prior to assembling ddPCR reactions lacking a restriction enzyme used the non-ITR assays in a duplex ddPCR milepost experiment to determine the genome integrity using linkage analysis.
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Fu, Donglai, and Yanhua Liu. "Remote Attestation on Behavioral Traces for Crowd Quality Control Based on Trusted Platform Module." Security and Communication Networks 2021 (April 26, 2021): 1–12. http://dx.doi.org/10.1155/2021/8859618.
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Behavioral traces of workers have emerged as a new evidence to check the quality of their produced outputs in crowd computing. Whether the evidence is trustworthy or not is a key problem during the process. Challenges will be encountered in addressing this issue, because the evidence comes from unknown or adversarial workers. In this study, we proposed an alternative approach to ensure trustworthy evidence through a hardware-based remote attestation to bridge the gap. The integrity of the evidence was used as the trustworthy criterion. Trusted Platform Module (TPM) was considered the trusted anchor inspired by trusted computing to avoid unreliable or malicious workers. The module carefully recorded and stored many workers’ behavioral traces in the storage measurement log (SML). Each item in the log was extended to a platform configuration register (PCR) by the occurrence sequence of each event. The PCR was a tamper-proof storage inside the TPM. The value of the PCR was also considered evidence together with the SML. The evidence was sent to the crowdsourcing platform with the TPM signature. The platform checked the integrity of the evidence by a series of operations, such as validating the signature and recomputing the SML hash. This process was designed as a remote attestation protocol. The effectiveness, efficiency, and security of the protocol were verified theoretically and through experiments based on the open dataset, WebCrowd25K, and custom dataset. Results show that the proposed method is an alternative solution for ensuring the integrity of behavioral traces.
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McCanless, Adair, Allison Hultgren, Cesar Escalante, Alyssa Ardt, and Rodrigo A. Valverde. "Effect of two digestive enzymes and pH on the dsRNA of endornaviruses of bell pepper and melon under in vitro conditions." Annals of Microbiology 69, no. 13 (2019): 1583–87. http://dx.doi.org/10.1007/s13213-019-01530-2.
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Abstract Purpose The objective of this investigation was to determine the in vitro effect of two common digestive enzymes, amylase and pepsin, and pH on the integrity of the RI dsRNA of bell pepper endornavirus (BPEV) and Cucumis melo endornavirus (CmEV) evaluated by gel electrophoresis and reverse-transcription PCR (RT-PCR). Methods We conducted experiments on the in vitro effect of two common digestive enzymes, amylase and pepsin, and pH on the structural integrity of the replicative intermediate (RI) dsRNA of bell pepper endornavirus (BPEV) and Cucumis melo endornavirus (CmEV), evaluated by gel electrophoresis and reverse-transcription polymerase chain reaction. Result The effect of the amylase, pepsin, and pH treatments on the dsRNA of both viruses was similar. Amylase did not appear to affect the structural integrity of the dsRNA. In contrast, gel electrophoresis analysis of pepsin-treated dsRNA samples showed an abnormal electrophoretic migration and evidence of partial dsRNA degradation. DsRNAs from both fruits were partially degraded when exposed to a pH value of 2.0 and completely degraded at a pH value of 1.0. Conclusion The results of this investigation suggest that when exposed to pepsin and pH values lower than 2.0, the RI of BPEV and CmEV lose their structural integrity. Therefore, when consuming endornavirus-infected bell pepper or melon, our digestive organs are exposed to both fragmented and full RI dsRNA of these two viruses.
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Mughal, Kiran, Mohammad Pervez Mughal, Muhammad Umar Farooq, Muhammad Qaiser Saleem, and Rodolfo Haber Guerra. "Helical Milling of CFRP/Ti6Al4V Stacks Using Nano Fluid Based Minimum Quantity Lubrication (NF-MQL): Investigations on Process Performance and Hole Integrity." Materials 16, no. 2 (2023): 566. http://dx.doi.org/10.3390/ma16020566.
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The structural components in the aeronautical industry require CFRP/Ti6Al4V stacks to be processed together, which results in poor hole integrity due to the thermal properties of the materials and challenges related to processability. These challenges include quality variation of the machined holes because of the limitations in process properties. Therefore, a novel solution through helical milling is investigated in the study using nano fluid based minimum quantity lubrication (NF-MQL). The analysis of variance shows, for Ti6Al4V, eccentricity (PCR = 28.56%), spindle speed (Ti) (PCR = 42.84%), and tangential feed (PCR = 8.61%), and for CFRP, tangential feed (PCR = 40.16%), spindle speed (PCR = 28.75%), and eccentricity (PCR = 8.41%) are the most significant parameters for diametric error. Further on, the rise in the circularity error is observed because of prolonged tool engagement at a higher value of tangential feed. Moreover, the surface roughness of Ti was reduced with an increasing percentage of MoS2 in the lubricant. The spindle speed (37.37%) and lubricant (45.76%) have a potential influence on the processing temperature, as evident in the analysis of variance. Similarly, spindle speed Ti (61.16%), tangential feed (23.37%), and lubrication (11.32%) controlled flank wear, which is critical to tool life. Moreover, the concentration of MoS2 decreased edge wear from ~105 µm (0.5% concentration) to ~70 µm (1% concentration). Thorough analyses on process performance in terms of hole accuracy, surface roughness, processing temperature, and tool wear are carried out based on the physical science of the process for cleaner production. The NF-MQL has significantly improved process performance and hole integrity.
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Frazer, Zoe, Changyoung Yoo, Manveer Sroya, et al. "Effect of Different Proteinase K Digest Protocols and Deparaffinization Methods on Yield and Integrity of DNA Extracted From Formalin-fixed, Paraffin-embedded Tissue." Journal of Histochemistry & Cytochemistry 68, no. 3 (2020): 171–84. http://dx.doi.org/10.1369/0022155420906234.
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DNA extracted from formalin-fixed, paraffin-embedded tissue sections is often inadequate for sequencing, due to poor yield or degradation. We optimized the proteinase K digest by testing increased volume of enzyme and increased digest length from the manufacturer’s protocol using 54 biospecimens, performing the digest in centrifuge tubes. Doubling the quantity of proteinase K resulted in a median increase in yield of 96%. Applying the optimized proteinase K protocol to sections deparaffinized on microscope slides generated a further increase in yield of 41%, but only at >50,000 epithelial tumor cells/section. DNA yield now correlated with (χ2 = 0.84) and could be predicted from the epithelial tumor cell number. DNA integrity was assayed using end point multiplex PCR (amplicons of 100–400 bp visualized on a gel), quantitative PCR (qPCR; Illumina FFPE QC Assay), and nanoelectrophoresis (DNA Integrity Numbers [DINs]). Generally, increases in yield were accompanied by increases in integrity, but sometimes qPCR and DIN results were conflicting. Amplicons of 400 bp were almost universally obtained. The process of optimization enabled us to reduce the percentage of samples that failed published quality control thresholds for determining amenability to whole genome sequencing from 33% to 7%.
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Padhi, Bhaja K., Manjeet Singh, Marianela Rosales, Guillaume Pelletier, and Sabit Cakmak. "A PCR-based quantitative assay for the evaluation of mRNA integrity in rat samples." Biomolecular Detection and Quantification 15 (May 2018): 18–23. http://dx.doi.org/10.1016/j.bdq.2018.02.001.
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Bohan, Amy E., Katelyn N. Purvis, Jason T. Sawyer, Werner G. Bergen, and Terry D. Brandebourg. "Sampling Adipose and Muscle Tissue following Post-Harvest Scalding Does Not Affect RNA Integrity or Real-Time PCR Results in Market Weight Yorkshire Pigs." Foods 11, no. 12 (2022): 1741. http://dx.doi.org/10.3390/foods11121741.
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Improving production efficiency while enhancing pork quality is pivotal for strengthening sustainable pork production. Being able to study both gene expression and indices of pork quality from the same anatomical location of an individual animal would better enable research conducted to study relationships between animal growth and carcass merit. To facilitate gene expression studies, adipose and muscle tissue samples are often collected immediately following exsanguination to maximize RNA integrity, which is a primary determinant of the sensitivity of RNA-based assays, such as real-time PCR. However, collecting soft tissue samples requires cutting through the hide or skin. This leaves the underlying tissue exposed during scalding, poses possible food safety issues, and potentially confounds pork quality measures. To overcome these limitations, the effect of tissue sample timing post-harvest on RNA integrity, real-time PCR results, and pork quality measurements was investigated by sampling subcutaneous adipose tissue and longissimus thoracis et lumborum muscle immediately following either exsanguination, scalding, or chilling. Sampling time did not affect RNA quality, as determined by the RNA integrity number of RNA samples purified from either adipose (RIN; p > 0.54) or muscle tissue (p > 0.43). Likewise, the sampling time did not influence the results of real-time PCR analysis of gene expression when comparing RNA samples prepared from adipose or muscle tissue immediately following either exsanguination or scalding (p > 0.92). However, sampling tissue prior to scalding resulted in a greater visual color score (p < 0.001) and lesser L* (p < 0.001) and b* (p < 0.001) values without impacting the 24 h pH (p < 0.41). These results suggested that if both RNA-based assays and meat quality endpoints are to be performed at the same anatomical location on an animal, tissue sampling to facilitate RNA-based assays should occur at a time point immediately following scalding. These findings demonstrated that sampling of adipose and muscle tissue can be delayed until after scalding/dehairing without decreasing the RNA integrity or altering the results of real-time PCR assays, while doing so was associated with little impact on measures of pork quality.
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Grote, Hans Jürgen, Viola Schmiemann, Mario Sarbia, and Alfred Böcking. "DNA Extraction from Bronchial Aspirates for Molecular Cytology: Which Method to Take?" Analytical Cellular Pathology 25, no. 2 (2003): 83–88. http://dx.doi.org/10.1155/2003/354796.
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Objective: To date, there are only few systematic reports on the quality of DNA extracted from routine diagnostic cytologic specimens. It was the aim of the present study to evaluate the ability of 50% ethanol/2% carbowax (Saccomanno fixative) to preserve bronchial secretions with high quality genomic DNA as well as to compare different DNA extraction methods.Methods: DNA was extracted from 45 bronchial aspirates by four different extraction protocols. Beside DNA yield, DNA quality with regard to purity, integrity, and PCR success rate were investigated.Results: No fragmentation of sample DNA due to the fixative was detected. It was preserved as high molecular weight DNA. DNA yield, purity, and integrity were dependent on the DNA extraction method to some extend. Irrespective of the DNA extraction method the PCR success rate for amplification of β‐globin gene fragments (268, 536, and 989 bp) was 100%. Conclusions: A fixative containing 50% ethanol/2% carbowax preserves high quality DNA which is well suited for PCR‐based assays regardless of the extraction protocol used. The selection of the DNA extraction protocol has to be adjusted to the circumstances of application.
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Ren, Sai, Xiaodong Ren, Haiqin Guo, et al. "Concentration and integrity indexes of urine cell-free DNA as promising biomarkers for early lung cancer diagnosis." Personalized Medicine 18, no. 2 (2021): 129–39. http://dx.doi.org/10.2217/pme-2020-0019.
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Aim: To explore the role of urine cell-free DNA (ucfDNA) concentration and integrity indexes as potential biomarkers for lung cancer diagnosis. Materials & methods: Quantitative real-time PCR targeting Arthrobacter luteus ( ALU) repeats at three size fragments ( ALU-60, 115 and 247 bp) was performed in 55 lung cancer patients and 35 healthy individuals. Results: ucfDNA concentration and integrity indexes were significantly higher in lung cancer patients than in healthy controls. The area under the receiver operating characteristic curve for differentiating patients with stage I/II from healthy controls by ALU fragments concentration were 0.856, 0.909 and 0.932, respectively. In addition, the ucfDNA integrity indexes in patients with lymph node metastasis were significantly higher than in patients with non-metastatic. Conclusion: ucfDNA concentration and integrity indexes could serve as promising biomarkers for lung cancer diagnosis.
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Wehmas, Leah C., Charles E. Wood, Brian N. Chorley, Carole L. Yauk, Gail M. Nelson, and Susan D. Hester. "Enhanced Quality Metrics for Assessing RNA Derived From Archival Formalin-Fixed Paraffin-Embedded Tissue Samples." Toxicological Sciences 170, no. 2 (2019): 357–73. http://dx.doi.org/10.1093/toxsci/kfz113.
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Abstract Formalin-fixed paraffin-embedded (FFPE) tissues provide an important resource for toxicogenomic research. However, variability in the integrity or quality of RNA obtained from archival FFPE specimens can lead to unreliable data and wasted resources, and standard protocols for measuring RNA integrity do not adequately assess the suitability of FFPE RNA. The main goal of this study was to identify improved methods for evaluating FFPE RNA quality for whole-genome sequencing. We examined RNA quality metrics conducted prior to RNA-sequencing in paired frozen and FFPE samples with varying levels of quality based on age in block and time in formalin. RNA quality was measured by the RNA integrity number (RIN), a modified RIN called the paraffin-embedded RNA metric, the percentage of RNA fragments >100–300 nucleotides in size (DV100–300), and 2 quantitative PCR-based methods. This information was correlated to sequencing read quality, mapping, and gene detection. Among fragmentation-based methods, DV and PCR-based metrics were more informative than RIN or paraffin-embedded RNA metric in determining sequencing success. Across low- and high-quality FFPE samples, a minimum of 80% of RNA fragments >100 nucleotides (DV100 > 80) provided the best indication of gene diversity and read counts upon sequencing. The PCR-based methods further showed quantitative reductions in amplifiable RNA of target genes related to sample age and time in formalin that inform input quantity of FFPE RNA for sequencing. These results should aid in screening and prioritizing archival FFPE samples for retrospective analyses of gene expression.
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Pacheco, Julia Iracema Moura, Ketlen Beatriz Alves dos Anjos, Isabela Vaz Silva, et al. "Comparison of two affordable DNA extraction methods for molecular detection of Salmonella isolates from broiler farm’s boot swabs." Research, Society and Development 12, no. 1 (2023): e28312139618. http://dx.doi.org/10.33448/rsd-v12i1.39618.
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There are many extraction methods available to purify bacterial DNA. PCR efficiency can vary depending on the quantity and quality of the template DNA used. This study evaluated the efficiency, quality, cost and rapidness of two in-house protocols, silica particles and Chelex-100 resin, for the extraction of twenty Salmonella isolates from boot swabs. The aim of this experiment was to compare the extraction methods for Salmonella spp. detection. DNA extraction was performed for each method and subjected to real-time PCR amplification. The amounts and integrity of DNA were determined using spectrophotometry and agarose gel electrophoresis, and the efficiency measured with real-time PCR. Limit of Detection (LOD) was determined with serial dilutions of a S. Typhimurium reference strain resulting in linear regression coefficients of R 2=0.992 (efficiency 119.31) for silica and R 2=0.958 (efficiency 93.33) for Chelex, with LOD of 10 -4 for both. Silica particles method resulted in higher DNA yield, 85.01 ± 59.11 compared to 50.74 ± 12.95 and 260/280 ratio, 1.77 ± 0.02 versus 1.63 ± 0.13. DNA integrity was superior using silica, gel showed a unique band higher than 2.000 bp, while Chelex-100 was imperceptive or degraded. Regarding PCR results, the mean quantification cycle (Cq) for silica was 17.08 ± 0.73 and Chelex-100, 17.64 ±0.56 (suggesting lower DNA values). Results showed that both methods were effective for the DNA extraction of Salmonella, once PCR resulted positive for all samples, efficiency was higher for silica. Chelex-100 resin was performed in less time at a lower cost.
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Palmirotta, Raffaele, Maria Laura De Marchis, Giorgia Ludovici, et al. "Impact of Preanalytical Handling and Timing for Peripheral Blood Mononuclear Cells Isolation and RNA Studies: The Experience of the Interinstitutional Multidisciplinary BioBank (BioBIM)." International Journal of Biological Markers 27, no. 2 (2012): 90–98. http://dx.doi.org/10.5301/jbm.2012.9235.
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Multicenter studies and biobanking projects require blood transportation from the participating center to a central collection or diagnostic laboratory. The impact of time delays between venous blood collection and peripheral blood mononuclear cells (PBMC) isolation prior to RNA extraction may affect the quality and quantity of isolated nucleic acids for genomic applications. Thus, standard operating procedure (SOP) optimization for the treatment of biological samples before RNA extraction is crucial in a biological repository. In order to define SOPs for whole blood preservation prior to RNA extraction, we sought to determine whether different blood storage times (0, 3, 6, 10, 24, and 30 hours) prior to PBMCs isolation and storage at –80°C, could affect the quality and quantity of extracted RNA. After spectrophotometric quantification, the quality and integrity of RNA were assessed by agarose gel electrophoresis, RNA integrity number and real time-PCR (RT-PCR). Across the different time points we did not observe significant differences within the first 24 hours of blood storage at room temperature, while a significant loss in RNA yield and integrity was detected between 24 and 30 hours. We conclude that time delays before PBMCs isolation prior to RNA extraction may have a significant impact on downstream molecular biological applications.
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Koentjoro, Maharani Pertiwi, Adyan Donastin, and Endry Nugroho Prasetyo. "A SIMPLE METHOD OF DNA EXTRACTION OF MYCOBACTERIUM TUBERCULOSIS FROM SPUTUM CULTURES FOR SEQUENCING ANALYSIS." African Journal of Infectious Diseases 15, no. 2s (2021): 19–22. http://dx.doi.org/10.21010/ajid.v15i2s.2.
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Background: Concern has been raised about DNA extraction from Mycobacterium tuberculosis due to its complex procedure. This study demonstrates a simple and fast DNA extraction method of mycobacterial genome to subsequent molecular investigation, such as Polymerization Chain Reaction (PCR) amplification, with species-specific primers and sequencing. Materials and Methods: Total DNA was isolated from M. tuberculosis cultured by using boil method. DNA was evaluated via measures of DNA quantity and quality (absorbance at 230, 260 and 280 nm), DNA integrity (electrophoresis). Molecular tests were tested namely PCR and sequencing. Conclusions: The quality of DNA obtained is acceptable for PCR and sequencing analysis. These findings demonstrate that the method used is inexpensive and suitable for minimum infrastructure facilities.
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Phillips, Jamie E., Stephen McCune, Corinne R. Fantz, Julia Engstrom-Melnyk, and John C. Osiecki. "Assay Integrity of a PCR Influenza Point-of-Care Test Remains Following Artificial System Contamination." Journal of Applied Laboratory Medicine 4, no. 3 (2019): 422–26. http://dx.doi.org/10.1373/jalm.2018.028639.
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Ekuni, Daisuke, James D. Firth, and Edward E. Putnins. "RNA integrity and in situ RT-PCR in dento-alveolar tissues after microwave accelerated demineralisation." Archives of Oral Biology 51, no. 2 (2006): 164–69. http://dx.doi.org/10.1016/j.archoralbio.2005.06.010.
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Lakpour, Niknam, Azadeh Mirfeizollahi, Shirin Farivar, et al. "The Association of Seminal Plasma Antioxidant Levels and Sperm Chromatin Status with Genetic Variants ofGSTM1andGSTP1(Ile105Val and Ala114Val) in Infertile Men with Oligoasthenoteratozoospermia." Disease Markers 34, no. 3 (2013): 205–10. http://dx.doi.org/10.1155/2013/635091.
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In this study we aimed to examine the effects of genetic variants ofGSTM1andGSTP1(Ile105Val and Ala114Val) on GST activity, seminal oxidative stress and sperm chromatin status in infertile men with oligoasthenoteratozoospermia (OAT). The study population (n= 121) consisted of 95 infertile men with OAT and 26 controls with normozoospermia. Multiplex polymerase chain reaction (PCR) and polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) methods were utilized to detect the aforesaid genetic variants. We measured GST activity and total antioxidant capacity (TAC) of seminal plasma by spectrophotometry. Sperm chromatin integrity and maturity were assessed using toluidine blue and chromomycin A3(CMA3-positive sperm) staining, respectively. The analysis showed that subgroups of GSTM1 null and GSTP1 C/T+T/T genotypes in comparison with GSTM1 present and GSTP1 wild type (C/C) genotypes did not have statistically significant differences in both OAT or normozoospermic men considering sperm concentration and motility, percentage of CMA3-positive sperm, seminal plasma TAC, sperm chromatin integrity and GST activity. Thus, the findings of our study suggest that there are no significant associations betweenGSTM1andGSTP1polymorphisms and sperm parameters at conventional or at molecular levels including OS status, sperm chromatin integrity or maturity in Iranian infertile men with OAT and normozoospermia. However, these polymorphisms could be related to the fertility status of the studied population but not evaluated in this study.
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Casadio, Valentina, Daniele Calistri, Samanta Salvi, et al. "Urine Cell-Free DNA Integrity as a Marker for Early Prostate Cancer Diagnosis: A Pilot Study." BioMed Research International 2013 (2013): 1–5. http://dx.doi.org/10.1155/2013/270457.
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Circulating cell-free DNA has been recognized as an accurate marker for the diagnosis of prostate cancer, whereas the role of urine cell-free DNA (UCF DNA) has never been evaluated in this setting. It is known that normal apoptotic cells produce highly fragmented DNA while cancer cells release longer DNA. We thus verified the potential role of UCF DNA integrity for early prostate cancer diagnosis. UCF DNA was isolated from 29 prostate cancer patients and 25 healthy volunteers. Sequences longer than 250 bp (c-Myc,BCAS1, andHER2) were quantified by real-time PCR to verify UCF DNA integrity. Receiver operating characteristic (ROC) curve analysis revealed an area under the curve of 0.7959 (95% CI 0.6729–0.9188). At the best cut-off value of 0.04 ng/μL, UCF DNA integrity analysis showed a sensitivity of 0.79 (95% CI 0.62–0.90) and a specificity of 0.84 (95% CI 0.65–0.94). These preliminary findings indicate that UCF DNA integrity could be a promising noninvasive marker for the early diagnosis of prostate cancer and pave the way for further research into this area.
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Zhang, Miaomiao, Xiaopeng Guo, Yue Gao, Dong Lu, and Wenjian Li. "Tumor Cell–Accelerated Senescence Is Associated With DNA-PKcs Status and Telomere Dysfunction Induced by Radiation." Dose-Response 16, no. 2 (2018): 155932581877152. http://dx.doi.org/10.1177/1559325818771527.
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Whether telomere structure integrity is related to radiosensitivity is not well investigated thus far. In this study, we investigated the relation between telomere instability and radiation-induced accelerated senescence. Partial knockdown of DNA-dependent catalytic subunit of protein kinase (DNA-PKcs) in human breast cancer cell line MCF-7 was established by small interfering RNA. Radiosensitivity of control and DNA-PKcs knockdown MCF-7 cells was analyzed by clonogenetic assay. Cell growth was measured by real-time cell electronic sensing. Senescence and apoptosis were evaluated by β-galactosidase histochemical staining and fluorescence-activated cell sorting, respectively. DNA damage was determined by long polymerase chain reaction (PCR). Telomere length and integrity were analyzed by real-time PCR and cytogenetic assay, respectively. DNA-PKcs knockdown MCF-7 cells were more sensitive to X-irradiation than control cells. Further investigation revealed that accelerated senescence is more pronounced than apoptosis in cells after radiation, particularly in DNA-PKcs knockdown cells. The cytogenetic assay and kinetics of DNA damage repair revealed that the role of telomere end-capping in DNA-PKcs, rather than DNA damage repair, was more relevant to radiosensitivity. To our knowledge, this is the first study to show that DNA-PKcs plays an important role in radiation-induced accelerated senescence via maintenance of telomere integrity in MCF-7 cells. These results could be useful for future understanding of the radiation-induced genome instability and its consequences.
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El-Shazly, Sherien F., Manal A. Eid, Hesham A. El-Sourogy, Gehan F. Attia, and Sherif A. Ezzat. "Evaluation of Serum Dna Integrity as a Screening and Prognostic Tool in Patients with Hepatitis C Virus-Related Hepatocellular Carcinoma." International Journal of Biological Markers 25, no. 2 (2010): 79–86. http://dx.doi.org/10.1177/172460081002500204.
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Background Hepatocellular carcinoma (HCC) is a common malignancy in Egypt due to the high frequency of hepatitis C virus (HCV) infection among the general population. Circulating free DNA is a potential molecular marker for the diagnosis and prognosis of malignant tumors. DNA released from apoptotic cells usually consists of short uniform fragments while DNA released from cancer cells is longer. The ratio of long DNA fragments to total DNA (DNA integrity) may be a potential marker for early detection of HCC and its progression in HCV patients. Methods Sera from 25 patients with HCV-related HCC, 25 patients with chronic HCV infection, and 15 healthy volunteers were examined for Alu repeats by quantitative real-time PCR (qPCR) using 2 sets of primers of 115 and 247 base pairs. DNA integrity was calculated as the ratio of 247-bp to 115-bp Alu fragments. Results Compared with healthy volunteers and HCV patients, significantly higher DNA integrity was found in HCC patients. DNA integrity was associated with tumor size, TNM stage, vascular invasion, lymph node involvement, and distant metastasis. DNA integrity had a higher sensitivity and specificity in discriminating HCC from HCV patients than total DNA. Patients with high DNA integrity had a significantly shorter overall survival and high DNA integrity was shown to be an independent prognostic factor for survival in HCV-related HCC. Conclusions DNA integrity is a promising molecular biomarker for detecting HCC in patients with chronic HCV infection; it reflects the progression and metastatic potential of the tumor, and high DNA integrity is associated with short overall survival in HCV-related HCC.
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Gurdal, Necla, Merdan Fayda, Nijat Alishev, et al. "Neoadjuvant volumetric modulated arc therapy in rectal cancer and the correlation of pathological response with diffusion-weighted MRI and apoptotic markers." Tumori Journal 104, no. 4 (2018): 266–72. http://dx.doi.org/10.5301/tj.5000702.
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Purpose: In this prospective observational study, we aimed to report the applicability and tolerability of neoadjuvant volumetric modulated arc therapy with simultaneous integrated boost (SIB-VMAT) and concurrent chemotherapy in patients with locally advanced rectal cancer (LARC), and to evaluate the correlation of pathological response with apparent diffusion coefficient (ADC) measurements on diffusion-weighted magnetic resonance imaging (DW-MRI) and apoptotic markers. Methods: The study enrolled 30 patients with T3 to T4 and/or N+ rectal cancer who preoperatively received SIB-VMAT and concurrent chemotherapy. Before and after the neoadjuvant treatment, apoptotic markers including the nucleosomes and cell-free DNA fragments in the serum samples were examined; DNA integrity was assessed by amplifying the ACTB gene; and the ADC measurements on the DW-MRI were analyzed. Results: No patients had acute or chronic grade III-IV toxicity. Pathologic complete response (pCR) was achieved in 8 patients (27%), while in 10 patients (33%) near-complete pathological response was obtained. Posttreatment ADC was significantly higher in patients with pCR compared with the others (1.28 vs. 1.10, p = 0.017). ROC curve analysis showed that posttreatment ADC values had a sensitivity of 75% and a specificity of 77.3% for distinguishing the patients with pCR from other responders. On the other hand, posttreatment DNA integrity values were revealed lower than the pretreatment values (p = 0.36). Also, the results revealed an insignificant increase in the posttreatment serum level of nucleosomes (p = 0.72). Conclusions: Neoadjuvant SIB-VMAT with concurrent chemotherapy was proved to be a feasible treatment regimen in LARC with tolerable side effects, and improved local control rate and pCR rate.
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Park, Noh Jin, Yang Li, Tianwei Yu, Brigitta MN Brinkman, and David T. Wong. "Characterization of RNA in Saliva." Clinical Chemistry 52, no. 6 (2006): 988–94. http://dx.doi.org/10.1373/clinchem.2005.063206.
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Abstract Background: We have previously shown that human mRNAs are present in saliva and can be used as biomarkers of oral cancer. In this study, we analyzed the integrity, sources, and stability of salivary RNA. Methods: We measured the integrity of salivary RNA with reverse transcription followed by PCR (RT-PCR) or RT-quantitative PCR (RT-qPCR). To study RNA entry sites into the oral cavity, we used RT-PCR analysis of salivary RNA from the 3 major salivary glands, gingival crevice fluid, and desquamated oral epithelial cells. We measured stability of the salivary β-actin mRNA by RT-qPCR of salivary RNA incubated at room temperature for different periods of time. We measured RNA association with other macromolecules by filtering saliva through pores of different sizes before performing RT-qPCR. To assess RNA–macromolecule interaction, we incubated saliva with Triton X-100 for different periods of time before performing RT-qPCR. Results: In most cases, we detected partial- to full-length salivary mRNAs and smaller amounts of middle and 3′ gene amplicons compared with the 5′. RNA was present in all oral fluids examined. Endogenous salivary β-actin mRNA degraded more slowly than exogenous β-actin mRNA, with half-lives of 12.2 and 0.4 min, respectively (P &lt;0.001). Salivary RNA could not pass through 0.22 or 0.45 μm pores. Incubation of saliva with Triton X-100 accelerated degradation of salivary RNA. Conclusions: Saliva harbors both full-length and partially degraded forms of mRNA. RNA enters the oral cavity from different sources, and association with macromolecules may protect salivary RNA from degradation.
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Cheng, S., Y. Chen, J. A. Monforte, R. Higuchi, and B. Van Houten. "Template integrity is essential for PCR amplification of 20- to 30-kb sequences from genomic DNA." Genome Research 4, no. 5 (1995): 294–98. http://dx.doi.org/10.1101/gr.4.5.294.
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Salvi, Samanta, Giorgia Gurioli, Filippo Martignano, et al. "Urine Cell-Free DNA Integrity Analysis for Early Detection of Prostate Cancer Patients." Disease Markers 2015 (2015): 1–6. http://dx.doi.org/10.1155/2015/574120.
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Introduction. The detection of tumor-specific markers in urine has paved the way for new early noninvasive diagnostic approaches for prostate cancer. We evaluated the DNA integrity in urine supernatant to verify its capacity to discriminate between prostate cancer and benign diseases of the urogenital tract.Patients and Methods. A total of 131 individuals were enrolled: 67 prostate cancer patients and 64 patients with benign diseases of the urogenital tract (control group). Prostate-specific antigen (PSA) levels were determined. Urine cell-free (UCF) DNA was isolated and sequences longer than 250 bp corresponding to 3 genes (c-MYC,HER2, andAR) were quantified by Real-Time PCR to assess UCF-DNA integrity.Results. UCF-DNA was quantifiable in all samples, while UCF-DNA integrity was evaluable in all but 16 samples. Receiver operating characteristic analysis showed an area under the curve of 0.5048 for UCF-DNA integrity and 0.8423 for PSA. Sensitivity was 0.58 and 0.95 for UCF-DNA integrity and PSA, respectively. Specificity was 0.44 and 0.69, respectively.Conclusions. UCF-DNA integrity showed lower accuracy than PSA and would not seem to be a reliable marker for early prostate cancer diagnosis. Despite this, we believe that UCF-DNA could represent a source of other biomarkers and could detect gene alterations.
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Deng, Yanan, Ziqi Qiao, Changping Zhou, et al. "Endothelial Myosin IIA Is Required for the Maintenance of Blood–Brain Barrier Integrity." Cells 13, no. 19 (2024): 1635. http://dx.doi.org/10.3390/cells13191635.
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Brain endothelial cells (ECs) are essential elements of the blood–brain barrier (BBB), maintaining its integrity through both paracellular junctions and transcellular transport systems. Myosin IIA, a multifunctional protein, plays a significant role in various cellular processes, including cytoskeletal maintenance, cell division, and signal transduction. While Myosin IIA has been implicated in bleeding and ischemic stroke, its role in regulating BBB integrity under physiological conditions remains unclear. In this study, we investigated the impact of Myosin IIA deficiency on BBB integrity using intravenous tracer injections and models of epilepsy. Flow cytometry, Western blot, and real-time PCR were employed to isolate brain cells and assess changes in protein and mRNA levels. Additionally, immunofluorescence staining and electron microscopy were used to explore alterations in protein expression and the structure of BBB. Our results demonstrate that endothelial Myosin IIA deficiency increased BBB permeability and exacerbated symptoms in BBB-related diseases. Mechanistically, we found that Myosin IIA modulates β-catenin transcription and protein interactions. The overexpression of β-catenin in brain endothelial Myosin IIA deficiency mice improved BBB integrity and reduced disease severity. This study establishes Myosin IIA as a critical regulator of BBB integrity and suggests new therapeutic targets for vascular diseases.
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Wong, Blenda CK, Rossa WK Chiu, Nancy BY Tsui, et al. "Circulating Placental RNA in Maternal Plasma Is Associated with a Preponderance of 5′ mRNA Fragments: Implications for Noninvasive Prenatal Diagnosis and Monitoring." Clinical Chemistry 51, no. 10 (2005): 1786–95. http://dx.doi.org/10.1373/clinchem.2005.052340.
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Abstract Background: The molecular characteristics of placental RNA circulating in maternal plasma are unknown. We investigated the integrity of circulating placental RNA in maternal plasma and tested the relevance of plasma RNA integrity for noninvasive prenatal diagnosis. Methods: Six different placental transcripts and mRNA of the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were quantified for the 5′ and 3′ regions in maternal plasma by 1-step real-time reverse transcription-PCR (RT-PCR) assays. This quantitative strategy was validated by 2-step RT-PCR and serial dilution experiments. The rates of detection by the 5′ and 3′ assays for the β-subunit of human chorionic gonadotropin (βhCG) were assessed in maternal plasma samples collected from different gestational periods. Results: For 5 of the 7 genes, the plasma mRNA concentrations measured by the 5′ amplicons were significantly higher than those measured by the corresponding 3′ amplicons. Every transcript under study demonstrated a higher rate of detection in the 5′ assay than in the 3′ assay in maternal plasma. In particular, the detection rate of βhCG mRNA in maternal plasma was increased throughout gestation when the 5′ assay was used. Conclusions: Circulating placental RNA is associated with a preponderance of 5′ mRNA fragments in maternal plasma. Apart from its intrinsic biological interest, this information could have important implications for the development of new assays targeting fetal RNA markers for noninvasive prenatal diagnosis and monitoring.
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Baert, Leen, Christiane E. Wobus, Els Van Coillie, Larissa B. Thackray, Johan Debevere, and Mieke Uyttendaele. "Detection of Murine Norovirus 1 by Using Plaque Assay, Transfection Assay, and Real-Time Reverse Transcription-PCR before and after Heat Exposure." Applied and Environmental Microbiology 74, no. 2 (2007): 543–46. http://dx.doi.org/10.1128/aem.01039-07.
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ABSTRACT The correlation between the detection of murine norovirus 1 RNA by real-time reverse transcription-PCR and the infectivity by plaque assay before and after heat exposure (80°C) was examined. No correlation was found in the current study. Moreover, heat inactivation had a much stronger detrimental effect on virus infectivity than on the integrity of the viral genome.
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Dieser, Markus, Andreas Nocker, John C. Priscu, and Christine M. Foreman. "Viable microbes in ice: application of molecular assays to McMurdo Dry Valley lake ice communities." Antarctic Science 22, no. 5 (2010): 470–76. http://dx.doi.org/10.1017/s0954102010000404.
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AbstractThe permanent ice covers of the McMurdo Dry Valley lakes, Antarctica, are colonized by a diverse microbial assemblage. We collected ice cores from Lakes Fryxell, Hoare and Bonney. Propidium monoazide (PMA) was used in combination with quantitative PCR (qPCR) and denaturing gradient gel electrophoresis (DGGE) to examine membrane integrity of prokaryotes in these extreme environments. PMA selectively penetrates cells with compromised membranes and modifies their DNA resulting in the suppression of PCR amplification. Our results based on analysis of 16S rRNA genes demonstrate that despite the hostile conditions of the Dry Valleys, the permanent ice covers of the lakes support a ‘potentially viable’ microbial community. The level of membrane integrity, as well as diversity, was higher in samples where sediment was entrapped in the ice cover. Pronounced differences in the fraction of cells with intact and compromised cell membranes were found for Lake Fryxell and east lobe of Lake Bonney, both expressed in differences in DGGE banding patterns and qPCR signal reductions. Limitations in the ability to distinguish between intact or compromised cells occurred in samples from Lake Hoare and west lobe of Lake Bonney due to low DNA template concentrations recovered from the samples.
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Veazey, Janelle, Timothy J. Chapman, Timothy R. Smyth, Sara E. Hillman, Sophia I. Eliseeva, and Steve N. Georas. "Protein Kinase D: A new target in the fight against flu?" Journal of Immunology 204, no. 1_Supplement (2020): 245.13. http://dx.doi.org/10.4049/jimmunol.204.supp.245.13.
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Abstract Summary Antivirals targeting host proteins may slow development of viral resistance and work against multiple viruses. The serine/threonine kinase protein kinase D (PKD) is one promising target as others have shown inhibiting PKD restricts rhinovirus and hepatitis in cell culture. We show inhibiting PKD restricts Influenza A virus (IAV) in cell culture and in mice. Methods The competitive PKD inhibitor, CRT, was given to A549, 16HBE cells and mice, prior to or during stimulation with dsRNA or IAV. Pulmonary barrier integrity was quantified by assaying total protein leak into the lumen (inside/out leak), and loss of 4kDa FITC-dextran out of the lumen (outside/in leak). Inflammatory cytokine levels were determined via ELISA/multiplex and mRNA levels via RT-PCR. Transcription factor activity was assayed via luciferase reporter system. Viral load was determined via RT-PCR of viral M protein. For in vivo IAV experiments, mice were given CRT/vehicle post-infection with lethal dose IAV (PR/8) and morbidity assessed by weight loss. Results Conclusion PKD inhibition promotes barrier integrity, lowers transcription of potentially pathogenic pro-inflammatory cytokines, and restricts viral replication, highlighting the potential of CRT as a novel anti-viral therapeutic.
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Zeng, Mu-Zi, Wei Zhou, Shan-Shan Wen, et al. "Identification and Functional Insights of Knickkopf Genes in the Larval Cuticle of Leptinotarsa decemlineata." Insects 15, no. 8 (2024): 623. http://dx.doi.org/10.3390/insects15080623.
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The Colorado potato beetle (Leptinotarsa decemlineata) is a major pest of potato crops. While Knickkopf (Knk) genes are essential for insect cuticle formation, their roles in pests like L. decemlineata remain unclear. This study aims to identify and characterize Knk genes in L. decemlineata and explore their functions in larval development and cuticle integrity. We used genomic and transcriptomic databases to identify LdKnk-family genes, validated through RT-PCR and RACE. Gene expression was analyzed at various developmental stages and tissues using qRT-PCR. RNA interference (RNAi) and Transmission electron microscopy (TEM) were applied to determine the functional roles of these genes. Four LdKnk-family genes were identified. Spatio-temporal expression analysis indicated significant gene expression during larval molting and pupal stages, especially in the epidermis. RNAi experiments showed that silencing LdKnk and LdKnk3-5′ led to reduced larval weight, cuticle thinning, and increased mortality, while LdKnk3-FL knockdown caused abnormal cuticle thickening and molting disruptions. LdKnk2 knockdown increased epicuticle and endocuticle thickness without visible phenotypic changes. The study highlights the essential roles of LdKnk-family genes in maintaining cuticle structure and integrity, suggesting their potential as targets for RNAi-based pest control.
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Guerra Naranjo, Cesar David, Tanira Alessandra Silveira Aguirre, and Ana Paula Guedes Frazzon. "DNARS: A safe, environmentally friendly and high-quality DNA extraction method suitable for various biological samples." Polo del Conocimiento 9, no. 10 (2024): 900–918. http://dx.doi.org/10.23857/pc.v9i10.8152.
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The functional and interactional relationships between nucleic acids and proteins form the foundational framework for molecular biology studies. In this context, obtaining high-quality nucleic acids is a crucial step for successful downstream applications. This study aimed to develop a cost-effective, eco-friendly, and versatile DNA extraction method that yields high-quality DNA from diverse biological samples. We utilized three types of borosilicate-based recycled laboratory glass as a silica source, combined with sodium iodide as a chaotropic agent, creating an efficient system for cell lysis and DNA extraction. Quality control was performed by assessing the concentration and purity of the extracted DNA using spectrophotometry, and the results were compared to those obtained with a commercial kit. DNA integrity was evaluated via agarose gel electrophoresis. To verify the suitability of the extracted DNA for downstream applications, we conducted 16S rRNA and ITS PCR amplifications. Our findings demonstrated that our DNA extraction method produced significantly higher yields, better purity, and greater integrity compared to the commercial kit. Moreover, the extracted DNA was readily applicable in PCR-based procedures, confirming the method's effectiveness for molecular biology applications.
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Park, Seong Kook, Woo Jeong Lee, and Young Il Yang. "Organ Culture at the Air–Liquid Interface Maintains Structural and Functional Integrities of Inflammatory and Fibrovascular Cells of Nasal Polyps." American Journal of Rhinology 21, no. 4 (2007): 402–7. http://dx.doi.org/10.2500/ajr.2007.21.3050.
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Background Both inflammatory and fibrovascular cells play an important role in development of nasal polyps (NPs). In this study, we have developed a culture system to maintain structural and functional integrities of submucosal cells in vitro. Methods NP tissue was cultured on a gelatin sponge at air–liquid (AL) interface or was cultured in submerging. Tissues were analyzed for survival, structural integrity, and vascular endothelial growth factor (VEGF) expression. Results Most cells of NPs cultured in submerging died within 3 days. In culture at the AL interface, epithelium as well as submucosa kept structural integrity during the culture period. RT-PCR and immunohistochemistry showed that submucosa cells displayed VEGF expression, which is a major inducer of angiogenesis and edema of NPs. Conclusion Our study is the first demonstration that organ culture of NPs at the AL interface retains integrity of both epithelium and submucosa and this culture method possibly will be used to study the pathogenesis of NPs.
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Mostofinejad, Zahra, Md Akheruzzaman, Md Abu Bakkar Siddik, Presheet Patkar, Nikhil V. Dhurandhar, and Vijay Hegde. "Antidiabetic E4orf1 protein prevents hepatic steatosis and reduces markers of aging-related cellular damage in high fat fed older mice." BMJ Open Diabetes Research & Care 9, no. 1 (2021): e002096. http://dx.doi.org/10.1136/bmjdrc-2020-002096.
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IntroductionOlder age is associated with greater prevalence of hyperinsulinemia, type 2 diabetes, and fatty liver disease. These metabolic conditions and aging are bidirectionally linked to mitochondrial dysfunction and telomere attrition. Although effectively addressing these conditions is important for influencing the health and the lifespan, it is particularly challenging in older age. We reported that E4orf1, a protein derived from human adenovirus Ad36, reduces hyperinsulinemia, improves glucose clearance, and protects against hepatic steatosis in younger mice exposed to high fat diet (HFD). Here, we tested if E4orf1 will improve glycemic control, liver fat accumulation, mitochondrial integrity, and reduce telomere attrition in older mice.Research design and methodsWe used 9-month-old mice that inducibly expressed E4orf1 in adipose tissue and non-E4orf1 expressing control mice. Mice were maintained on a 60% (kcal) HFD for 20 weeks and glycemic control was determined by intraperitoneal glucose tolerance test at week 20. Following 20 weeks of HF-feeding, mice were sacrificed and liver tissues collected to determine the expression of aging genes using qRT-PCR based RT2 Profiler PCR array.ResultsCompared with the control mice, E4orf1 significantly improved glycemic control and reduced hepatic steatosis and fibrosis. Additionally, E4orf1 maintained markers of mitochondrial integrity and telomere attrition.ConclusionE4orf1 has the potential to improve glycemic control in older mice, and the improvement persists even after longer term exposure. E4orf1 expression also maintains mitochondrial integrity and telomere attrition, thus delaying age-associated diseases. This provides strong evidence for therapeutic utility of E4orf1 in improving age-associated metabolic and cellular changes that occur with aging in humans.
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Relova, Damarys, Lester J. Pérez, Liliam Ríos, et al. "Short communication: Stability and integrity of classical swine fever virus RNA stored at room temperature." Spanish Journal of Agricultural Research 15, no. 3 (2017): e05SC03. http://dx.doi.org/10.5424/sjar/2017153-10776.
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Worldwide cooperation between laboratories working with classical swine fever virus (CSFV) requires exchange of virus isolates. For this purpose, shipment of CSFV RNA is a safe alternative to the exchange of infectious material. New techniques using desiccation have been developed to store RNA at room temperature and are reported as effective means of preserving RNA integrity. In this study, we evaluated the stability and integrity of dried CSFV RNA stored at room temperature. First, we determined the stability of CSFV RNA covering CSFV genome regions used typically for the detection of viral RNA in diagnostic samples by reverse transcription-polymerase chain reaction (RT-PCR). To this end, different concentrations of in vitro-transcribed RNAs of the 5’-untranslated region and of the NS5B gene were stored as dried RNA at 4, 20, and 37oC for two months. Aliquots were analyzed every week by CSFV-specific quantitative real-time RT-PCR. Neither the RNA concentration nor the storage temperature did affect CSFV RNA yields at any of the time evaluated until the end of the experiment. Furthermore, it was possible to recover infectious CSFV after transfection of SK-6 cells with dried viral RNA stored at room temperature for one week. The full-length E2 of CSFV was amplified from all the recovered viruses, and nucleotide sequence analysis revealed 100% identity with the corresponding sequence obtained from RNA of the original material. These results show that CSFV RNA stored as dried RNA at room temperature is stable, maintaining its integrity for downstream analyses and applications.
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Stellino, Chiara, Gaël Hamot, Camille Bellora, Johanna Trouet, and Fay Betsou. "Preanalytical robustness of blood collection tubes with RNA stabilizers." Clinical Chemistry and Laboratory Medicine (CCLM) 57, no. 10 (2019): 1522–29. http://dx.doi.org/10.1515/cclm-2019-0170.
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Abstract Background Efficient blood stabilization is essential to obtaining reliable and comparable RNA analysis data in preclinical operations. PAXgene (Qiagen, Becton Dickinson) and Tempus (Applied Biosystems, Life Technologies) blood collection tubes with RNA stabilizers both avoid preanalytical degradation of mRNA by endogenous nucleases and modifications in specific mRNA concentrations by unintentional up- or down-regulation of gene expression. Methods Sixteen different preanalytical conditions were tested in PAXgene and Tempus blood samples from seven donors: different mixing after collection, different fill volumes and different 24-h transport temperature conditions after collection. RNA was extracted by column-based methods. The quality of the extracted RNA was assessed by spectrophotometric quantification, A260/A280 purity ratio, RNA Integrity Number (Agilent Bioanalyzer), miRNA quantative real time polymerase chain reaction (qRT-PCR) on two target miRNAs (RNU-24 and miR-16), mRNA quality index by qRT-PCR on the 3′ and 5′ region of the GAPDH gene, and the PBMC preanalytical score, based on the relative expression levels of the IL8 and EDEM3 coding genes. Results When PAXgene RNA and Tempus blood collection tubes were used following the manufacturers’ instructions, there was no statistically or technically significant difference in the output RNA quality attributes. However, the integrity of the RNA extracted from Tempus collection tubes was more sensitive to fill volumes and effective inversion, than to storage temperature, while the integrity of RNA extracted from PAXgene collection tubes was more sensitive to effective inversion and storage temperature than to fill volumes. Conclusions Blood collection tubes with different RNA stabilizers present different robustness to common preanalytical variations.
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Duanmu, Luyang, Youji Shen, Ping Gong, Hao Zhang, Xiangkai Meng, and Yuanhua Yu. "Constant Pressure-Regulated Microdroplet Polymerase Chain Reaction in Microfluid Chips: A Methodological Study." Micromachines 15, no. 1 (2023): 8. http://dx.doi.org/10.3390/mi15010008.
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Digital polymerase chain reaction (PCR) technology in microfluidic systems often results in bubble formation post-amplification, leading to microdroplet fragmentation and compromised detection accuracy. To solve this issue, this study introduces a method based on the constant pressure regulation of microdroplets during PCR within microfluidic chips. An ideal pressure reference value for continuous pressure control was produced by examining air solubility in water at various pressures and temperatures as well as modeling air saturation solubility against pressure for various temperature scenarios. Employing a high-efficiency constant pressure device facilitates precise modulation of the microfluidic chip’s inlet and outlet pressure. This ensures that air solubility remains unsaturated during PCR amplification, preventing bubble precipitation and maintaining microdroplet integrity. The device and chip were subsequently utilized for quantitative analysis of the human epidermal growth factor receptor (EGFR) exon 18 gene, with results indicating a strong linear relationship between detection signal and DNA concentration within a range of 101–105 copies/μL (R2 = 0.999). By thwarting bubble generation during PCR process, the constant pressure methodology enhances microdroplet stability and PCR efficiency, underscoring its significant potential for nucleic acid quantification and trace detection.
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Sugiana, Fenny Aulia, Henni Widyowati, Muhammad Ali Warisman, Suryani Suryani, and Desriani Desriani. "Low cost and comprehensive pork detection in processed food products with a different food matrix." Indonesian Journal of Biotechnology 23, no. 1 (2018): 21. http://dx.doi.org/10.22146/ijbiotech.32372.
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The adulteration of processed beef-based meat products with pork is a sensitive issue in Indonesia. In this study, we developed a detection method for the low cost identification of pork in processed meat products. We used the cost-efficient Taq DNA polymerase, DreamTaq Green PCR master mix (2x), and duplex PCR method to recognize pork simultaneously with 18S rRNA detection. A positive control containing a pork gene inserted into pGEM®-T easy was prepared, along with a negative control. The results of the duplex PCR were used to assess its specificity, detection limit, and its ability to recognize pork in processed meat products with a different food matrix. 18S rRNA detection was for confirming DNA integrity of DNA extracted from the processed food, while the positive control confirmed that the reagents were working well and the negative control confirmed a non-contamination problem. Following this, the duplex PCR was optimized and the optimum concentration primer for duplex PCR detection was found to be 3 µm for pork and 0.2 µm for 18S rRNA. As little as 3.125 ng of the DNA template could be used to detect whether a sample contained pork. Duplex PCR is a simple, fast, sensitive, specific, and low cost method of detecting pork in processed meat products.
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Ragland, Natalie H., Emily L. Miedel, and Robert W. Engelman. "PCR Prevalence of Murine Opportunistic Microbes and their Mitigation by Using Vaporized Hydrogen Peroxide." Journal of the American Association for Laboratory Animal Science 58, no. 2 (2019): 208–15. http://dx.doi.org/10.30802/aalas-jaalas-18-000112.
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Exposing immunodeficient mice to opportunistic microbes introduces risks of data variability, morbidity, mortality, and the invalidation of studies involving unique human reagents, including the loss of primary human hematopoietic cells, patient-derived xenografts, and experimental therapeutics. The prevalence of 15 opportunistic microbes in a murine research facility was determined by yearlong PCR-based murine and IVC equipment surveillance comprising 1738 specimens. Of the 8 microbes detected, 3 organisms— Staphylococcus xylosus, Proteus mirabilis, and Pasteurella pneumotropica biotype Heyl—were most prevalent in both murine and IVC exhaust plenum specimens. Overall, the 8 detectable microbes were more readily PCR-detectable in IVC exhaust airways than in murine specimens, supporting the utility of PCR testing of IVC exhaust airways as a component of immunodeficient murine health surveillance. Vaporized hydrogen peroxide (VHP) exposure of IVC equipment left unassembled (that is, in a 'static-open' configuration) did not eliminate PCR detectable evidence of microbes. In contrast, VHP exposure of IVC equipment assembled 'active-closed' eliminated PCR-detectable evidence of all microbes. Ensuring data integrity and maintaining a topographically complex immunodeficient murine research environment is facilitated by knowing the prevalent opportunistic microbes to be monitored and by implementing a PCR-validated method of facility decontamination that mitigates opportunistic microbes and the risk of invalidation of studies involving immunodeficient mice.
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Umetani, Naoyuki, Joseph Kim, Suzanne Hiramatsu, et al. "Increased Integrity of Free Circulating DNA in Sera of Patients with Colorectal or Periampullary Cancer: Direct Quantitative PCR for ALU Repeats." Clinical Chemistry 52, no. 6 (2006): 1062–69. http://dx.doi.org/10.1373/clinchem.2006.068577.
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Abstract Background: Cell-free DNA circulating in blood is a candidate biomarker for malignant tumors. Unlike uniformly truncated DNA released from apoptotic nondiseased cells, DNA released from dead cancer cells varies in size. We developed a novel method to measure the ratio of longer to shorter DNA fragments (DNA integrity) in serum as a potential biomarker for patients with colorectal cancer (CRC) or periampullary cancers (PACs). Methods: Sera from 32 patients with CRC (3 stage I, 14 stage II, 6 stage III, and 9 stage IV patients), 19 patients with PACs (2 stage I, 9 stage II, 1 stage III, and 7 stage IV patients), and 51 healthy volunteers were assessed by quantitative real-time PCR of ALU repeats (ALU-qPCR) with 2 sets of primers (115 and 247 bp) amplifying different lengths of DNA. We used serum directly as a template for ALU-qPCR without DNA purification. DNA integrity was determined as ratio of qPCR results of 247-bp ALU over 115-bp ALU. Results: ALU-qPCR had a detection limit of 0.01 pg of DNA. Eliminating DNA purification reduced technical artifacts and reagent/labor costs. Serum DNA integrity was significantly increased for stage I/II and III/IV CRC and stage I/II and III/IV PACs (P = 0.002, P = 0.006, P = 0.022, and P &lt;0.0001, respectively). ROC curves for detecting CRC and PACs had areas under the curves of 0.78 and 0.80, respectively. Conclusions: Direct ALU-qPCR is a robust, highly sensitive, and high-throughput method to measure serum DNA integrity. DNA integrity is a potential serum biomarker for detection and evaluation of CRC and PACs.