Absolute Quantitation of mRNA and DNA Copy Number in Different Nutritional States: A Methodological Approach in Primer Design, mRNA Copy Number and Discrimination between Highly Homologous Gene Families
Authors: Rote Paula , Shirota Leticia Akina , Sharma Katyayini , Hardwick James P 
Published: November 2023

Numerous gene expression studies report the relative quantitation of gene expression by quantitative polymerase chain reaction (qPCR) compared to a control sample and reference gene. Although this method is suitable for animal studies in one tissue, relative quantitation of gene expression in humans is severely limited by the absence of a control sample or a normalizing reference due to variations in expression related to gender, race, ethnicity and dietary preferences. Furthermore, relative gene expression between different tissues is impossible to gauge due to inherent differences in the levels of reference gene expression.Absolute quantitation of the number of mRNA molecule overcomes the limitations of relative mRNA quantitation by not requiring a control sample or a reference gene to normalize mRNA expression, and allows for comparison of mRNA expression under various conditions 1) between different tissues, 2) between polymorphic alleles, and 3) within highly homologous gene of gene families.Here we present a method for quantifying mRNA or DNA copy number per nanogram of cDNA/DNA using Rutledge's linear regression efficiency (LRE) program, with calibration standards confirmed by droplet digital PCR, and copy number verified through dual priming oligonucleotides. For highly homologous gene sequences, we utilized poly‐inosine primers capable of selectively amplifying as few as one base pair mismatch. To highlight the usefulness of this method in biological samples, we determined the number of mRNA molecules for members of the CYP4 gene family. Since many members of CYP4a subfamily have over 95% nucleotide sequence similarity, we used dual priming oligonucleotide, poly‐inosine primers to selectively detect expression of CYP4a10, CYP4a31, and CYP4a32 mRNA, which share greater than 98% sequence similarity.We compared this modified LRE method with mass action kinetic (MAK) model for quantification of lambda DNA standards and found good correlation between these models. Copy number determined by digital PCR, though very precise in the low copy number range, did not produce reliable copy numbers above 10,000 and, in biological samples, did not show the specificity with poly‐inosine primers seen in qPCR with LRE. With this modified LRE method, we observed massive differences in the expression of CYP4a mRNA during fasting and starvation, detecting less than 100 mRNA molecules to over 1 million mRNA molecules for different members of the CYP4 family. We also observed a sexual dimorphic response between males and females in CYP4a gene expression in response to fasting.This methodological approach to the quantitation of absolute number of mRNA molecules, variation in DNA gene copies, and determination of polymorphic allele expression provides a critical basic and clinical tool to quantitate changes in mRNA and DNA number of molecules in oncogene gene duplication in cancer, viral load infections, changes in the molecular and metabolic pathways in numerous human diseases, and human susceptibility to drugs and toxins in the field of pharmacogenetics.