Student Wiki on methodology

This Wiki is intended to collectively make the point on methodologies employed in research papers we analyze during the course. "Writers" are students who wish to contribute to a specific subject. Before contributing, please add your name in the "Writers group choice". When initiating a contribution, please indicate your name in brackets.


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This page contains the links to the nine official subjects, which are the same in the Choice.

To contribute, go to the correct page by clicking on the description here in the index, then click EDIT and contribute. At the end, please save.

 IMPORTANT !!!

Please do not make extensive cut-and-paste: it s useless, anybody can go to the source you use and read it.  Read the texts, digest, and make a short résumé. If you wih you can include link(s) to the source(s).

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Transcriptome: special techniques, RNA-Seq, GRO-Seq, CAGE, others.

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RNA-seq

Transcriptome Analysis is the study of the transcriptome, of the complete set of RNA transcripts that are produced by the genome, under specific circumstances or in a specific cell, using high-throughput methods.

Transcriptome analysis by next-generation (RNA-seq) sequencing allows investigation of a transcriptome at unsurpassed resolution, detecting both coding and regulatory transcripts, like siRNA and lncRNA. One major benefit is that RNA-seq is independent of a priori knowledge on the sequence under investigation, thereby also allowing analysis of poorly characterized species. 

Brief outline of the workflow

1. bulk RNA is extracted from the sample and the desired RNA is selected (sample preparation)

2. the selected RNA is copied into stable double-stranded copy DNA (library construction)

3. the ds cDNA is then sequenced using various sequencing methods

4. the sequences obtained can are aligned to reference genome sequences, available in data banks, to identify which genes are transcribed. This type of analysis provides a quantification of the expression levels for the transcribed genes. Alternatively, RNA-seq can be used to identify alternative splicing, novel transcripts, and fusion genes, following a new transcript discovery approach.

RNA-seq workflow

The complete workflow of RNA-seq consist of: (1) experimental design; (2) sample and library preparation; (3) sequencing; and (4) data analysis. You will find a general explanation of each step in the following video.


For a deeper understanding of the RNA-seq technology and application follow the links:

https://www.intechopen.com/books/applications-of-rna-seq-and-omics-strategies-from-microorganisms-to-human-health/rna-seq-applications-and-best-practices 

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4648566/