Sensitive and Low-Bias Transcriptome Sequencing Using Agarose PCR.

Area of Science:

• Molecular Biology
• Genomics
• Neuroscience

Background:

• Transcriptome sequencing is crucial for single-cell research but limited by amplification bias in trace RNA library preparation.
• Existing amplification methods introduce bias, hindering accurate analysis of limited biological samples.

Purpose of the Study:

• To develop a novel amplification method to improve sensitivity and reduce bias in transcriptome sequencing of trace RNA.
• To evaluate the efficacy of incorporating low-melting-point agarose into PCR for enhanced RNA amplification.

Main Methods:

• Low-melting-point agarose was added to PCR amplification reactions to leverage its molecular crowding and cross-linked structure.
• The agarose PCR method was applied to single-cell transcriptome sequencing of mouse brain tissue microregions from Parkinson's disease models.
• Performance was compared against traditional in-tube PCR for sensitivity and homogeneity.

Main Results:

• Agarose PCR demonstrated superior performance compared to in-tube PCR, yielding results closer to unamplified data.
• Sensitivity of the amplification reaction significantly increased with agarose addition, and homogeneity improved approximately twofold.
• A notable 11% sensitivity improvement was achieved for detecting Parkinson's disease-associated genes in spatial transcriptomic studies.

Conclusions:

• Agarose PCR offers a new, efficient, and homogeneous amplification tool for minute biological samples.
• This method significantly enhances transcriptome library sequencing, particularly for spatial transcriptomics and low-input applications.
• The technique holds broad potential for advancing single-cell and spatial transcriptomic analyses in various research fields.