Enrichment of mRNA and Bisulfite-mRNA Library Preparation for Next-Generation Sequencing

Szu-Ying Chen; Po-Hsien Huang

doi:10.3791/65352

Journal

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Biology

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Enrichment of mRNA and Bisulfite-mRNA Library Preparation for Next-Generation Sequencing

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Biology

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JoVE Journal Biology

Enrichment of mRNA and Bisulfite-mRNA Library Preparation for Next-Generation Sequencing

1,013 Views

•

06:57 min

•

July 07, 2023

DOI:

10.3791/65352-v

DOI:

10.3791/65352-v

•

06:57 min

July 07, 2023

•

5 Views

Szu-Ying Chen^1,2, Po-Hsien Huang^1,2

¹Institute of Basic Medical Sciences, College of Medicine,National Cheng Kung University, ²Department of Biochemistry and Molecular Biology, College of Medicine,National Cheng Kung University

Transcript

This research aims to study the role of RNA methylation, specifically 5-methylcytosine modification, in pancreatic cancer. The questions we want to answer are how upregulation or downregulation of certain RNA methyltransferase affects cancer cell phenotypes and also, through which RNA substrates. This study provides the comprehensive RNA m5C landscape at a single-base resolution with standardized equipment.

From this study using bisulfite RNA seq, the results links the association of RNA m5C modification with poor differentiation of cancer cells. This should pave the way for future studies in discerning passenger versus disease driver effects of specific m5C in the RNA transcripts. With this technique, we can identify the set of differentially-methylated RNAs that may participate in tumor progression.

Further, single molecule base experiments can provide a more detailed regulatory role of specific RNA m5C.

Summary

This protocol provides an easy-to-follow workflow to conduct poly(A) RNA purification, bisulfite conversion, and library preparation using standardized equipment for a biological sample of interest.