Whole blood RNA extraction efficiency contributes to variability in RNA sequencing data sets.

Area of Science:

• Molecular Biology
• Genomics
• Bioinformatics

Background:

• RNA extraction methodologies vary, leading to quantitative differences in recovered RNA.
• These variations can influence downstream RNA sequencing results, particularly for mRNA and lncRNA.
• Understanding these influences is crucial for accurate transcriptomic analysis.

Purpose of the Study:

• To evaluate how different blood RNA extraction methods affect mRNA and lncRNA sequencing outcomes.
• To determine the impact of total RNA quantity on transcript recovery and expression analysis.
• To investigate the relationship between transcript size and sequencing results across different extraction methods.

Main Methods:

• Analysis of three archived blood RNA sequencing datasets.
• Evaluation of RNA extraction methodologies' influence on mRNA and lncRNA recovery.
• Normalization of common transcripts and plotting of TPM counts and CV against transcript size.
• Regression analysis to assess relationships between transcript size, TPM counts, and CV.

Main Results:

• Lower total RNA recovery per ml of blood impacted the recovery of weakly expressed and short to midsized transcripts.
• Two datasets showed a decrease in TPM counts with increasing transcript size, while one dataset showed increased counts proportional to size.
• Transcript recovery variability across extraction methods altered the fundamental relationship between transcript size and TPM counts.

Conclusions:

• The quantity of total RNA recovered from whole blood significantly influences RNA sequencing results.
• Incomplete RNA extraction directly affects the recovery of mRNA and lncRNA transcripts.
• Differences in RNA extraction efficiency likely contribute to batch effects observed in sequencing data.