The emerging technique of microfluidic digital PCR (dPCR) offers a unique approach to real-time quantitative PCR for measuring nucleic acids that may be particularly suited for low-level detection. In this study, we evaluated the quantitative capabilities of dPCR when measuring small amounts (<200 copies) of DNA and investigated parameters influencing technical performance. We used various DNA templates, matrixes, and assays to evaluate the precision, sensitivity and reproducibility of dPCR, and demonstrate that this technique can be highly reproducible when performed at different times and when different primer sets are targeting the same molecule. dPCR exhibited good analytical sensitivity and was reproducible outside the range recommended by the instrument manufacturer; detecting 16 estimated targets with high precision. The inclusion of carrier had no effect on this estimated quantity, but did improve measurement precision. We report disagreement when using dPCR to measure different template types and when comparing the estimated quantities by dPCR and UV spectrophotometry. Finally, we also demonstrate that preamplification can impose a significant measurement bias. These findings provide an independent assessment of low copy molecular measurement using dPCR and underline important factors for consideration in dPCR experimental design.
Gene expression measurements detailing mRNA quantities are widely employed in molecular biology and are increasingly important in diagnostic fields. Reverse transcription (RT), necessary for generating complementary DNA, can be both inefficient and imprecise, but remains a quintessential RNA analysis tool using qPCR. This study developed a Transcriptomic Calibration Material and assessed the RT reaction using digital (d)PCR for RNA measurement. While many studies characterise dPCR capabilities for DNA quantification, less work has been performed investigating similar parameters using RT-dPCR for RNA analysis. RT-dPCR measurement using three, one-step RT-qPCR kits was evaluated using single and multiplex formats when measuring endogenous and synthetic RNAs. The best performing kit was compared to UV quantification and sensitivity and technical reproducibility investigated. Our results demonstrate assay and kit dependent RT-dPCR measurements differed significantly compared to UV quantification. Different values were reported by different kits for each target, despite evaluation of identical samples using the same instrument. RT-dPCR did not display the strong inter-assay agreement previously described when analysing DNA. This study demonstrates that, as with DNA measurement, RT-dPCR is capable of accurate quantification of low copy RNA targets, but the results are both kit and target dependent supporting the need for calibration controls.
Primary or premature ovarian insufficiency (POI) is the most common long-term complication experienced by girls and women with classic galactosemia; more than 80% and perhaps more than 90% are affected despite neonatal diagnosis and careful lifelong dietary restriction of galactose. In this review we explore the complexities of timing and detection of galactosemia-associated POI and discuss potential underlying mechanisms. Finally, we offer recommendations for follow-up care with current options for intervention.
We undertook a genomewide linkage study in a total of 353 affected sib pairs (ASPs) with schizophrenia. Our sample consisted of 179 ASPs from the United Kingdom, 134 from Sweden, and 40 from the United States. We typed 372 microsatellite markers at approximately 10-cM intervals. Our strongest finding was a LOD score of 3.87 on chromosome 10q25.3-q26.3, with positive results being contributed by all three samples and a LOD-1 interval of 15 cM. This finding achieved genomewide significance (P<.05), on the basis of simulation studies. We also found two regions, 17p11.2-q25.1 (maximum LOD score [MLS] = 3.35) and 22q11 (MLS = 2.29), in which the evidence for linkage was highly suggestive. Linkage to all of these regions has been supported by other studies. Moreover, we found strong evidence for linkage (genomewide P<.02) to 17p11.2-q25.1 in a single pedigree with schizophrenia. In our view, the evidence is now sufficiently compelling to undertake detailed mapping studies of these three regions.
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