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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 microarray-based...

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Updated: Jul 9, 2026

Purifying the Impure: Sequencing Metagenomes and Metatranscriptomes from Complex Animal-associated Samples

Purifying the Impure: Sequencing Metagenomes and Metatranscriptomes from Complex Animal-associated Samples

Published on: December 22, 2014

Optimising RNA extraction for paddy bulk soil samples for metatranscriptome sequencing.

Sumitra Sivaprakasam¹, Tan Yee Fan¹, Yukgehnaish Kumarasan¹

¹Centre of Excellence for Omics-Driven Computational Biodiscovery (COMBio), AIMST University, Bedong, Kedah, 08100, Malaysia.

BMC Research Notes

|July 5, 2025

Summary

This summary is machine-generated.

Optimizing RNA extraction from paddy soil using polyethylene glycol (PEG) precipitation significantly improved RNA yield and quality. This enhanced method is crucial for accurate microbiome analysis in plant growth studies.

Keywords:

Bulk soil Metatranscriptome sequencing Paddy RNA extraction

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Published on: July 1, 2022

Area of Science:

Microbiology
Molecular Biology
Plant Science

Background:

High-quality RNA extraction is essential for studying microbiome molecular dynamics in paddy plants.
Paddy soil's humic substances and clay content hinder RNA extraction, causing adsorption and low yields.
Existing RNA extraction methods require optimization for challenging soil environments.

Purpose of the Study:

To improve existing RNA extraction protocols for bulk soil samples from Malaysian paddy fields.
To optimize RNA extraction for highly pigmented and challenging paddy soil samples.
To evaluate the suitability of the optimized RNA for metatranscriptome sequencing.

Main Methods:

Evaluation of multiple published RNA extraction protocols.
Optimization of a selected protocol (Peng et al., 2018, method B3) using polyethylene glycol (PEG) precipitation (20% and 30%).
Assessment of RNA yield, purity (A260/A280), and integrity (RIN), followed by metatranscriptome sequencing.

Main Results:

The optimized protocol using 20% PEG precipitation effectively removed pigmentation, yielding pigment-free RNA (>100 ng/µl, integrity >7, A260/A280 of 2.02 ± 0.02).
Metatranscriptome sequencing and analysis (Trinity, BUSCO, Kraken2) confirmed superior RNA quality.
The 20% PEG method demonstrated higher bacterial read assignment, indicating effectiveness for downstream applications.

Conclusions:

The optimized RNA extraction protocol using 20% PEG precipitation is highly effective for paddy soil.
This method significantly enhances RNA quality and yield, overcoming challenges posed by soil contaminants.
The extracted RNA is suitable for robust microbial transcriptomic analyses and microbiome studies in paddy ecosystems.