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Methods in molecular exercise physiology
Published in Adam P. Sharples, James P. Morton, Henning Wackerhage, Molecular Exercise Physiology, 2022
Adam P. Sharples, Daniel C. Turner, Stephen Roth, Robert A. Seaborne, Brendan Egan, Mark Viggars, Jonathan C. Jarvis, Daniel J. Owens, Jatin G. Burniston, Piotr P. Gorski, Claire E. Stewart
RNA-sequencing, typically referred to as ‘RNA-seq’, is a thorough method for analysing all RNAs simultaneously. Unlike microarrays, RNA-seq does not require prior knowledge of the RNA sequence that is explored and, therefore, has the capacity to detect single nucleotide variants, alternate splicing, post-transcriptional modifications and silencing RNAs (also called micro-RNAs/miRNA), non-coding RNA (ncRNA), exon-intron boundaries and pre-mRNA via NGS technology. The present section will focus on the use of RNA-seq for quantifying gene expression, as this is the analysis that is becoming more frequently used in molecular exercise physiology. To undertake RNA-seq, sequencing ‘libraries’ are first created using the isolated RNA. As with RT-qPCR and microarrays, RNA is first isolated, and reverse transcribed to cDNA. The cDNA is then fragmented into small consistent sizes and sequencing ‘adaptors’ are added or ‘ligated’ to the ends of the cDNA fragments. These adaptors contain constant sequences that enable the sequencers to recognise where to start sequencing. NGS is then performed similar to that explained in the above sections. Prior to these crucial steps, the total RNA obtained from the muscle biopsy may be first treated to remove ribosomal RNA that makes up the majority (~90%) of extracted RNA. Therefore, if left untreated, sequencing data would be representative of predominantly rRNA rather than other RNAs of interest (e.g. pre-mRNA, mRNA, miRNA and ncRNA).
Cancer Informatics
Published in Trevor F. Cox, Medical Statistics for Cancer Studies, 2022
DNA and RNA sequencing can be carried out by various methods, for example there is SOLID sequencing, Ion Torrent sequencing, Illumina (Solexa) sequencing and others. The sequencing may be for the whole genome or just part of it. Every short-strand (fragment) of the PCR enhanced DNA/RNA is sequenced, with all the results being stored in a file. The read length of a fragment is the length of the sequence of the fragment. The fragment length is the average of the read lengths. The sequencing is not perfect, with errors creeping in, and so the sequencing process also produces a probability of an error, p, for each base in a sequence. The probability is transformed into a quality score, . For , , which might be used as a cut-off for poor quality, higher values being of acceptable quality. The files produced have various formats, for example, FASTQ files which have four lines for each sequence fragment, for example,
Genetic Basis of Neuromuscular Disorders
Published in Maher Kurdi, Neuromuscular Pathology Made Easy, 2021
RNA sequencing (RNA-Seq) of patient samples has recently emerged as a complementary technique with very promising diagnostic yield. It uses the capabilities of high-throughput sequencing methods to provide an insight into cellular transcriptome and total cellular content of RNAs including mRNA, rRNA, and tRNA. It can also tell us which genes are turned on, what are their levels of expression, and at what time they are activated. It specifically facilitates identification of spliced transcripts, post transcriptional modification, gene fusion, mutation/SNP changes, and also finds differences in gene expression between two or more conditions. The latter is called the differential expression process.
RNA-seq analysis identifies transcriptomic profiles associated with anal cancer recurrence among people living with HIV
Published in Annals of Medicine, 2023
Yuanfan Ye, Kevin J. Maroney, Howard W. Wiener, Olga A. Mamaeva, Anna D. Junkins, Greer A. Burkholder, Staci L. Sudenga, Mohd Khushman, Sameer Al Diffalha, Anju Bansal, Sadeep Shrestha
Identifying predictive biomarkers regarding therapeutic impacts and disease prognosis using RNA-sequencing (RNA-seq) based approaches has been widely applied in the field of cancer research [24]. While the use of this approach revealed significant coding and non-coding transcripts in various cancers [25], transcriptomic signatures in SCCA are lacking, specifically with treatment response outcomes. In this study, we characterized gene expression profiling in SCCA tumor tissues from PLWH to compare coding (mRNA) and non-coding (e.g. miRNA, IncRNA) transcripts that were differentially expressed among successful CRT versus recurrent SCCA patients. The transcriptomic biomarkers identified in this study can provide insights into the pathological processes driving observed treatment outcomes and need to be validated in larger cohorts in future studies to determine whether they can be accurately used to screen for early diagnosis of patients at high risk of having recurrent anal cancer.
UROSCAN and UROSCANSEQ: a large-scale multicenter effort towards translation of molecular bladder cancer subtypes into clinical practice – from biobank to RNA-sequencing in real time
Published in Scandinavian Journal of Urology, 2023
Fredrik Liedberg, Johan Abrahamsson, Carina Bernardo, Mats Bläckberg, Anders Edsjö, Markus Heidenblad, Christer Larsson, Gottfrid Sjödahl, Pontus Eriksson
The lack of complete coverage of biobanking and RNA-sequencing are limitations of the current effort, needing improvement during the coming years. We additionally plan to expand the infrastructure to include blood samples at routine intervals to test hypotheses related to circulating tumor cells and presence of ctDNA to identify individuals suitable for adjuvant checkpoint-inhibition after radical cystectomy [29], despite that the intention-to-treat analysis was negative in the original phase III trial [30]. To further strengthen the current research effort, we also extend an invitation to other hospitals in Sweden and the Nordic countries to join the UROSCANSEQ network. Additionally, to include sampling and subtyping of metastatic lesions would also increase the knowledge about bladder cancer biology. Finally, the single-sample classifiers for molecular subtyping need to comply with the recent in vitro diagnostic medical devices regulation (IVDR) (2017/746/EU) before broader clinical implementation is launched.
QPCTL regulates macrophage and monocyte abundance and inflammatory signatures in the tumor microenvironment
Published in OncoImmunology, 2022
Kaspar Bresser, Meike E. W. Logtenberg, Mireille Toebes, Natalie Proost, Justin Sprengers, Bjorn Siteur, Manon Boeije, Lona J. Kroese, Ton N. Schumacher
RNA sequencing. RNA was extracted from the indicated frozen tissues using the RNeasy Mini Kit (Qiagen). Cell populations isolated by FACS were washed once in PBS, and subsequently lysed in RLT buffer (Qiagen). Whole-transcriptome sequencing samples were prepared with the TruSeq Stranded mRNA Kit (Illumina). Paired-end 50 bp sequencing was performed on a NovaSeq 6000 system (S1 flowcell, Illumina), obtaining an average of 18 × 106 reads per sample. Reads were aligned to the pre-built GRCm38 genome_snp_tran reference using HISAT2,50 and transcript counts were obtained using an in-house generated pipeline (GenSum, https://github.com/NKI-GCF/gensum). Differential gene expression analysis was performed using the edgeR package.51 Network analysis was performed using the stringDB database, applying the igraph package for visualization.