A simple analytical and experimental procedure for selection of reference genes for reverse-transcription...

Area of Science:

• Molecular Biology
• Genomics
• Animal Science

Background:

• Cellular activity variations impact RNA concentration, affecting gene mRNA abundance analysis via RT-qPCR.
• Reference genes are crucial for normalizing gene expression data, but their selection requires careful validation.

Purpose of the Study:

• To propose an alternative protocol for assessing reference gene invariance in porcine mammary tissue.
• To utilize mammary RNA and DNA concentrations as correction factors for improved accuracy.

Main Methods:

• Biopsies from sows at different physiological stages (gestation, lactation, postweaning).
• RT-qPCR quantification of 7 potential reference genes and 1 candidate gene.
• Linear mixed model analysis incorporating RNA and DNA concentrations as covariates.
• Comparison with geNorm analysis for reference gene selection.

Main Results:

• GAPDH and MRPL39 showed unchanged mRNA abundance after accounting for RNA and DNA concentration variations.
• geNorm identified MTG1, MRPL39, and VAPB as top reference genes.
• Normalization of the target gene SLC7A1 yielded similar results regardless of the reference gene selection method.

Conclusions:

• The proposed protocol effectively assesses reference gene expression invariance by correcting for tissue RNA and DNA concentration changes.
• This method offers a reliable alternative for selecting suitable reference genes in RT-qPCR studies.
• Accurate reference gene selection is vital for valid gene expression profiling in dynamic physiological states.