A Complete Guide to Whole Transcriptome Sequencing

What is whole transcriptome?

Whole transcriptome refers to the collection of all transcript information from a specific tissue or cell transcribes in a specific state, including mRNA, small RNA, lncRNA, circRNA and other coding and non-coding RNAs. Transcriptomics is the basis of scientific research and the significance of its research is well known. In recent years, in addition to miRNAs and hot lncRNAs, the ceRNA analysis has begun to emerge, becoming a dazzling star in the scientific research community.

Why do we do whole transcriptome sequencing?

To answer this question, let's talk about what is ceRNA.

ceRNA (competing endogenous RNA) is not a new RNA molecule, but an entirely new mode of gene expression regulation. The ceRNA hypothesis reveals a new mechanism of interaction between RNAs.

ceRNA - competitive endogenous RNA

There is a region on the mRNA or ncRNA that can be bound by the miRNA, ie, an MREs functional element, and an RNA molecule containing homologous MREs can competitively bind to the miRNA. It is known that miRNAs can cause gene silencing by binding to target genes, ceRNAs can regulate the expression of target genes by competitively binding miRNAs.

At present, the study of a single mRNA or ncRNA can no longer meet scientific research needs. Combining various RNA information for joint ceRNA analysis and exploring its potential regulatory network mechanisms have become a powerful tool for interpreting biological phenomena.

How to do whole transcriptome sequencing?

Whole-transcriptome sequencing is the application of technology. By constructing two library-small RNA libraries and a de novo ribosome-specific library, four types of RNA (miRNA, lncRNA, mRNA, circRNA) information can be analyzed at one time.

3.1 Analysis content

The whole transcriptome analysis content is more comprehensive, in addition to the four kinds of RNA standard analysis, it also contains a joint analysis of ceRNA with advanced analysis content.

Analysis Process of Whole Transcriptome Sequencing

Figure: Analysis Process of Whole Transcriptome Sequencing

ceRNA interaction regulation network

3.2 Research Ideas

Research Procedures of Whole Transcriptome Sequencing

1) Determine the research object

Controls & Treatments; Health & Diseases; Cancer & Paracancetic Tissues...

2) Obtain 4 RNA data using RNA-seq

3) Screening for differentially expressed mRNA, lncRNA, miRNA, circRNA

4) Construction of ceRNA regulatory networks and screening of ceRNA-mRNA regulatory pairs

5) QRT-PCR verifies the correlation of expression levels

6) Verification of ceRNA regulatory relationships

a. Verify the effect of ceRNAs on target genes from protein levels, RNA levels, etc.;

b. Analysis of miRNA binding sites, ceRNA-miRNA regulatory relationships;

7) Functional verification

a. Cellular levels: cell proliferation, apoptosis and cycle changes, tumor metastasis, etc.;

b. Molecular level: Western Blot and immunohistochemistry, siRNA interference/overexpression vector, immunoprecipitation (IP), etc.;

c. Animal experiments: construction of animal models, tumor models;

d. Clinical level: detection of changes in animal phenotypes and biochemical indicators, postoperative recovery.

3.3 Case Analysis

Practice is the sole criterion for testing truth. Two articles published recently in Nature communications explored its regulatory mechanism, with lncRNA and circRNA as the main research points and combined with mRNA and miRNA data for ceRNA analysis.

1. The lncRNA-mediated sponge regulatory network in prostate cancer

Studies have shown that lncRNA can be used as a miRNA sponge to competitively bind to mRNA. In this paper, two sp-lncRNAs were screened by , and it was determined that PTEN is its regulatory gene, and it acts through miRNAs, indicating that the lncRNA-mediated sponge regulatory network plays an important role in the regulation of cancer.

2. Mechanism of miRNA sponge interaction in circRNA

In this paper, RNA-seq technology was used to screen differential circRNAs, circHIPK3 was identified as the target molecule, and identification, generation, and intracellular localization were studied. Through miRNA binding site analysis combined with multiple experimental verification, it was shown that circHIPK3 can function as a miRNA sponge.

Reference

1. Differentially expressed lncRNAs and miRNAs with associated ceRNA networks in aged mice with postoperative cognitive dysfunction, https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5593532/

2. RNA: Circular RNAs as miRNA sponges, https://www.nature.com/articles/nrm3557

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