Quantitative transcriptomics using designed primer-based amplification.

Area of Science:

• Molecular Biology
• Genomics
• Developmental Biology

Background:

• Accurate RNA quantification from limited samples is crucial for understanding gene expression.
• Existing RNA-sequencing methods face challenges with low RNA input and preserving transcript abundance.
• Ribosomal RNA often dominates sequencing libraries, masking low-abundance transcripts.

Purpose of the Study:

• To develop a novel RNA-sequencing strategy for sensitive and accurate transcriptomic analysis.
• To enable the study of gene expression from minute amounts of RNA.
• To identify novel transcripts and understand cell fate specification during early development.

Main Methods:

• Developed a Designed Primer-based RNA-sequencing (DP-seq) strategy using defined heptamer primers.
• Optimized DP-seq for amplifying transcripts from as low as 50 picograms of mRNA.
• Applied DP-seq to study lineage segregation in embryonic stem cell cultures.

Main Results:

• DP-seq reproducibly amplified the majority of expressed transcripts from limited mRNA, preserving relative abundance.
• Achieved a dynamic range of over five orders of magnitude in RNA concentrations.
• Successfully suppressed amplification of ribosomal transcripts by over 70%.
• DP-seq identified novel low-abundance transcripts, including those indicative of cell fate specification prior to germ layer segregation.

Conclusions:

• DP-seq is a powerful tool for sensitive and accurate RNA-sequencing from limited samples.
• The strategy effectively preserves transcript abundance and reduces bias from highly expressed transcripts.
• DP-seq provides novel insights into early mammalian embryogenesis and cell fate determination.