Use of blood RNA sequencing (RNA-seq) as a splicing analysis tool for clinical interpretation of variants of uncertain significance (VUSs) found via whole-genome and exome sequencing can be difficult for genes that have low expression in the blood due to insufficient read count coverage aligned to specific genes of interest. Here, we present a short amplicon reverse transcription-polymerase chain reaction(RT-PCR) for the detection of genes with low blood expression. Short amplicon RT-PCR, is designed to span three exons where an exon harboring a variant is flanked by one upstream and one downstream exon. We tested short amplicon RT-PCRs for genes that have median transcripts per million (TPM) values less than one according to the genotype-tissue expression database. Median TPM values of genes analyzed in this study are SYN1 = 0.8549, COL1A1 = 0.6275, TCF4 = 0.4009, DSP = .2894, TTN = 0.2851, COL5A2 = 0.1036, TERT = 0.04452, NTRK2 = 0.0344, ABCA4 = 0.00744, PRPH = 0, and WT1 = 0. All these genes show insufficient exon-spanning read coverage in our RNA-seq data to allow splicing analysis. We successfully detected all genes tested except PRPH and WT1. Aberrant splicing was detected in SYN1, TCF4, NTRK2, TTN, and TERT VUSs. Therefore, our results show short amplicon RT-PCR is a useful alternative for the analysis of splicing events in genes with low TPM in blood RNA for clinical diagnostics.
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http://dx.doi.org/10.1002/humu.24378 | DOI Listing |
mSphere
December 2024
Department of Pharmacology & Therapeutics, Faculty of Medicine and Health Sciences, McGill University, Montréal, Québec, Canada.
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View Article and Find Full Text PDFAm J Obstet Gynecol
December 2024
Department of Epidemiology and Public Health, University of Maryland School of Medicine, Baltimore, MD, USA; Institute for Genome Sciences, University of Maryland School of Medicine, Baltimore, MD, USA. Electronic address:
Background: Vaginal lubricants are commonly used during sexual activity and clinical procedures such as transvaginal ultrasound (TVUS). Epidemiologic and laboratory studies indicate hyperosmolal water-based lubricants may disrupt the vaginal microbiota, particularly the beneficial Lactobacillus spp. These bacteria play a critical role in protecting against sexually transmitted infection acquisition and other adverse gynecologic and obstetric outcomes.
View Article and Find Full Text PDFWater Res X
January 2025
Great Lakes Institute for Environmental Research, University of Windsor, Windsor ON N9B 3P4, Canada.
UV disinfection is extensively used for wastewater disinfection and disinfection efficiency is commonly monitored using culture-based enumeration of . While culture-independent real-time quantitative polymerase chain reaction (qPCR) based methods are attractive due to faster turnaround and easier application, previous attempts with qPCR to monitor disinfection have been unsuccessful. In this study, the effect of UV irradiation on a pure culture was examined in collimated beam (CB) experiments and monitored using both a culturing technique and DNA damage quantified using both short amplicon (SA; <∼200 bp) qPCR and longer amplicon (LA; ∼500-bp) qPCR.
View Article and Find Full Text PDFFoods
November 2024
College of Food Science and Engineering, Bohai University, Jinzhou 121013, China.
This study aimed to ascertain the potential benefits of green radish polysaccharide (GRP) in treating alcoholic liver disease (ALD) in mice and explore its mechanism of action. Using biochemical analysis, high-throughput sequencing of gut microbiota, and gas chromatography-mass spectrometry to measure short-chain fatty acids (SCFAs) in feces, we found that GRP intervention significantly improved lipid metabolism and hepatic function in mice subjected to excessive alcohol intake. The GRP intervention reduced malondialdehyde levels by 66% and increased total superoxide dismutase levels by 22%, thereby mitigating alcohol-induced oxidative stress.
View Article and Find Full Text PDFmSystems
December 2024
Department of Biology, The Pennsylvania State University, University Park, Pennsylvania, USA.
Unlabelled: Phylogenetic marker gene sequencing is often used as a quick and cost-effective way of evaluating microbial composition within a community. While 16S rRNA gene sequencing (16S) is commonly used for bacteria and archaea, other marker genes are preferable in certain situations, such as when 16S sequences cannot distinguish between taxa within a group. Another situation is when researchers want to study cospeciation of host taxa that diverged much more recently than the slowly evolving 16S rRNA gene.
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