Blocking Abundant RNA Transcripts by High-Affinity Oligonucleotides during Transcriptome Library Preparation.

Area of Science:

• Molecular Biology
• Genomics
• Biotechnology

Background:

• RNA sequencing (RNA-seq) is a powerful tool for transcriptome analysis.
• Quantifying low-abundant RNA transcripts remains a challenge in RNA-seq due to competition with highly abundant species.
• Existing methods struggle to accurately measure transcripts present at low concentrations.

Purpose of the Study:

• To develop an accessible strategy for improving the detection of low-abundant RNA transcripts.
• To overcome the limitations of current RNA sequencing quantification methods.
• To enhance transcriptome-wide coverage and complexity analysis.

Main Methods:

• Development of a method utilizing high-affinity RNA-binding oligonucleotides.
• Application of blocking oligonucleotides to inhibit reverse transcription and PCR amplification of specific abundant transcripts.
• Testing the strategy across diverse RNA types (YRNAs, mitochondrial rRNAs, MALAT1) and sequencing protocols (small RNA, 3' end, long-read, single-cell).

Main Results:

• Demonstrated a significant reduction in the abundance of targeted high-abundance RNA transcripts.
• Achieved improved transcriptome coverage and complexity in sequencing libraries.
• Confirmed the efficiency, reproducibility, and specificity of the blocking strategy.
• Successfully applied the method to various biological samples and sequencing techniques.

Conclusions:

• The developed blocking oligonucleotide strategy is highly effective for enhancing low-abundant RNA detection.
• The method is easily integrated into existing RNA sequencing workflows without protocol modification.
• This approach offers a versatile solution for improving transcriptome analysis across different applications.