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RNA-seq

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Related Experiment Video

Updated: Jan 10, 2026

Optimization for Sequencing and Analysis of Degraded FFPE-RNA Samples

Optimization for Sequencing and Analysis of Degraded FFPE-RNA Samples

Published on: June 8, 2020

Pre-sequencing assessment of RNA-Seq library quality using real-time qPCR.

Kavya Kottapalli¹, Hsu Chao¹, Qi Jiang¹

¹Human Genome Sequencing Center, Baylor College of Medicine, Houston, TX, USA.

|November 26, 2025

Summary

This summary is machine-generated.

A new quantitative polymerase chain reaction (qPCR) assay accurately predicts ribosomal RNA (rRNA) content in RNA sequencing libraries before sequencing. This cost-effective method ensures reliable transcriptome profiling by assessing rRNA depletion efficiency.

Keywords:

18S qPCR Illumina Oligo (dT) beads Poly A+RNA-Seq Total RNA depletion efficiency rRNA removal

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Published on: November 7, 2025

Area of Science:

Molecular Biology
Genomics
Biotechnology

Background:

Ribosomal RNA (rRNA) constitutes a major fraction of cellular RNA, necessitating its efficient removal for accurate transcriptome analysis via RNA sequencing (RNA-Seq).
Inefficient rRNA depletion in RNA-Seq library preparation can compromise the accurate sequencing of low-abundance messenger RNAs (mRNAs).
Current methods for assessing rRNA content pre-sequencing are often unreliable, costly, or lack scalability.

Purpose of the Study:

To develop and validate a scalable, cost-effective assay for quantifying rRNA depletion efficiency in RNA-Seq libraries prior to sequencing.
To establish a reliable method for predicting the percentage of rRNA reads in pre-sequencing libraries.

Main Methods:

Development of a real-time quantitative polymerase chain reaction (qPCR) assay targeting human 18S rRNA.
Optimization of qPCR efficiency using serial dilutions of Universal Human Reference (UHR) control.
Establishment of Ct thresholds using pilot data from 644 libraries.
Validation using 1748 human Total RNA-Seq and 445 Poly A+ RNA-Seq libraries.
Evaluation of mRNA enrichment performance using Oligo (dT) beads from four vendors.

Main Results:

The 18S rRNA qPCR assay demonstrated a strong correlation between pre-sequencing rRNA estimates and post-sequencing rRNA read percentages across diverse RNA-Seq library types.
The assay proved effective in evaluating the performance of different mRNA enrichment methods.

Conclusions:

The developed 18S rRNA qPCR assay provides a reliable, scalable, and cost-effective solution for assessing rRNA depletion efficiency in RNA-Seq libraries.
This assay enables researchers to predict and potentially mitigate issues related to rRNA contamination before undertaking expensive sequencing.
The method supports improved accuracy and efficiency in transcriptome profiling studies.