A method for simultaneous detection of small and long RNA biotypes by ribodepleted RNA-Seq

Area of Science:

• Molecular Biology
• Genomics
• Transcriptomics

Background:

• RNA sequencing (RNA-Seq) provides comprehensive transcriptome analysis.
• Simultaneous detection of diverse RNA species is crucial for understanding gene expression.
• Existing protocols may face challenges with specific tissue types or rRNA removal.

Purpose of the Study:

• To develop and validate a novel, streamlined RNA sequencing protocol.
• To enable simultaneous detection and quantification of both coding and non-coding transcripts.
• To improve RNA-Seq efficiency and consistency across different sample types.

Main Methods:

• Modification of the Ion Total RNA-Seq kit v2 protocol.
• Integration of the QIASeq FastSelect rRNA removal kit for efficient rRNA depletion.
• Application of the protocol to frozen mouse hippocampal and post-mortem human tissues.
• Validation using quantitative real-time PCR (qRT-PCR).

Main Results:

• Consistent sequencing library preparation from both high-integrity mouse and challenging human post-mortem tissues.
• Highly efficient rRNA removal (<1.5% in final library) using FastSelect.
• Identification and quantification of >30,000 unique transcripts, including coding and non-coding RNAs.
• Normalized sequencing read counts showed significant negative correlation with qRT-PCR Ct values, indicating accuracy.

Conclusions:

• The developed protocol accurately and consistently identifies and quantifies a wide range of transcripts simultaneously.
• Efficient rRNA depletion and minimized sample handling enhance its utility.
• This protocol is valuable for researchers investigating whole transcriptomes, particularly with challenging samples.