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RNA-seq

RNA-seq

RNA sequencing, or RNA-Seq, is a high-throughput sequencing technology used to study the transcriptome of a cell. Transcriptomics helps to interpret the functional elements of a genome and identify the molecular constituents of an organism. Additionally, it also helps in understanding the development of an organism and the occurrence of diseases.
Before the discovery of RNA-seq, microarray-based methods and Sanger sequencing were used for transcriptome analysis. However, while...

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Mapping RNA-RNA Interactions Globally Using Biotinylated Psoralen

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Standardization of RNA chemical mapping experiments.

Wipapat Kladwang¹, Thomas H Mann, Alex Becka

¹Department of Biochemistry, Stanford University , Stanford, California 94305, United States.

|April 29, 2014

Summary

Standardizing chemical RNA mapping methods like dimethyl sulfate (DMS) and SHAPE improves data accuracy. This approach corrects errors in sequencing and electrophoretic data, enabling precise RNA structure analysis.

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Area of Science:

Molecular Biology
Biochemistry
Bioinformatics

Background:

Chemical mapping provides insights into RNA structure.
Current data processing relies on ad hoc assumptions, limiting accuracy.
Standardization is needed for quantitative RNA mapping.

Purpose of the Study:

To develop and validate standardized methods for chemical RNA mapping.
To improve the accuracy of RNA structure determination using deep sequencing and electrophoretic data.
To establish rigorous corrections for overmodification, background, and ligation bias.

Main Methods:

Utilized simple dilutions and referencing standards (GAGUA hairpins).
Applied HiTRACE/MAPseeker analysis for data processing.
Standardized protocols for dimethyl sulfate (DMS), 2 -OH acylation (SHAPE), and carbodiimide measurements across six noncoding RNAs.

Main Results:

Achieved rigorous overmodification correction, background subtraction, and normalization.
Implemented ligation bias correction for deep sequencing data.
Identified new signatures for extrahelical bulges and DMS "hot spot" pockets, including tRNA A58.

Conclusions:

The proposed standardization is essential for accurate and quantitative RNA mapping.
Standardized methods enhance the reliability of RNA structure determination.
New signatures reveal finer details of RNA structural elements.