Developingmessenger RNA biomarkers: A workflow to characterise and identify transcript target sequences unaffected by alternative splicing for reproducible gene transcript quantification by reverse transcriptase quantitative polymerase chain reaction

Abstract

Most eukaryotic genes generate multiple messenger RNA (mRNA) transcript variants by alternative splicing. The incomplete annotation of gene transcripts in genomic databases can result in improper primer design, adversely affecting the reliability of gene expression measurements by reverse transcriptase quantitative polymerase chain reaction (RT-qPCR). Hence, we present aworkflow combining bioinformatics analyses, to select two to three evolutionarily conserved constitutive exons in rats, mice and humans as target sequences for PCR primer design, with experimental RT-PCR amplification and amplicon sequencing to confirm the expression and identity of gene transcript targets. The application of this workflow to the characterization of neurodevelopmental biomarker genes identified an unannotated exon in the rat Map2 gene, illustrating the importance of target sequence validation for the development of translational mRNA biomarkers for toxicological and biomedical studies.

Keywords

alternative splicing / amplicon sequencing / exon-exon junction / RT-qPCR / transcriptional biomarker

References

[1] DevonshireAS, Sanders R, WilkesTM, TaylorMS, FoyCA, HuggettJF. Application of next generation qPCR and sequencing platforms to mRNA biomarker analysis. Methods. 2013;59(1):89-100.

[2] BustinSA, BenesV, GarsonJ, et al. The need for transparency and good practices in the qPCR literature. Nat Methods. 2013;10(11):1063-1067.

[3] PanQ, ShaiO, LeeLJ, Frey BJ, BlencoweBJ. Deep surveying of alternative splicing complexity in the human transcriptome by high-throughput sequencing. Nat Genet. 2008;40(12):1413-1415.

[4] MoralesJ, PujarS, LovelandJE, et al. A joint NCBI and EMBL-EBI transcript set for clinical genomics and research. Nature. 2022;604(7905):310-315.

[5] AmaralP, Carbonell-Sala S, De La VegaFM, et al. The status of the human gene catalogue. Nature. 2023;622(7981):41-47.

[6] JiX, LiP, FuscoeJC, et al. A comprehensive rat transcriptome built from large scale RNA-seq-based annotation. Nucleic Acids Res. 2020;48(15):8320-8331.

[7] RuijterJM, Ruiz-Villalba A, van den HoffAJJ, GunstQD, Wittwer CT, van den HoffMJB. Removal of artifact bias from qPCR results using DNA melting curve analysis. FASEB J. 2019;33(12):14542-14555.

[8] PadhiBK, Pelletier G, ShwedPS. A bioinformatics workflow for the evaluation of RT-qPCR primer specificity: application for the assessment of gene expression data reliability in toxicological studies. Regul Toxicol Pharmacol. 2020;111:104575.

[9] FuGC, LinWC. Identification of gene-oriented exon orthology between human and mouse. BMC Genomics. 2012;13:S10. Suppl 1.

[10] PadhiBK, Rosales M, PelletierG. Perinatal methylmercury exposure perturbs the expression of Plp1 and Cnp splice variants in cerebellum of rat pups. Neurotoxicology. 2015;48:223-230.

[11] PadhiBK, SinghM, HuangN, Pelletier G. A PCR-based approach to assess genomic DNA contamination in RNA: application to rat RNA samples. Anal Biochem. 2016;494:49-51.

[12] PadhiBK, SinghM, PelletierG. A bioinformatics framework for targeted gene expression assay design: application to in vitro developmental neurotoxicity screening in a rat model. Regul Toxicol Pharmacol. 2022;133:105211.

[13] MazinPV, Khaitovich P, Cardoso-MoreiraM, KaessmannH. Alternative splicing during mammalian organ development. Nat Genet. 2021;53(6):925-934.

[14] KerachianMA, Azghandi M, ThieryJP. A proposal to improve gene expression analysis. Clin Transl Dis. 2022;2:e26.

RIGHTS & PERMISSIONS

2024 His Majesty the King in Right of Canada. Clinical and Translational Discovery published by JohnWiley & Sons Australia, Ltd on behalf of Shanghai Institute of Clinical Bioinformatics. Reproduced with the permission of the Minister of Health Canada.