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A simple method to assess group difference in RT-qPCR reference gene selection using GeNorm: The case of the placental sex

St-Pierre, Joey; Gregoire, Jean-Charles; Vaillancourt, Cathy . A simple method to assess group difference in RT-qPCR reference gene selection using GeNorm: The case of the placental sex In: Meeting of the International-Federation-of-Placenta-Associations (IFPA), August 30-September 02, 2017, Manchester, England.

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Résumé

Objectives:The reverse transcription quantitative polymerase chain re-action (RT-qPCR) is an efficient method to assess mRNA level in a variety ofsamples. Normalization with proper reference genes is a crucial step inobtaining accurate mRNA expression levels. GeNorm and NormFinder aretwo commonly used software packages that help selecting the bestreference genes based on their expression stability, but, however, GeNormdoes not take into account a group variable (such as sample sex) in itscalculation. Here, we demonstrate a simple calculation step to assess thevariability of such parameters by multiplying the GeNorm M value withthe difference of Cq values between groups.

Methods:To test this, we have used PrimePCR plates (Bio-Rad) with 28reference gene candidates to analyse 20 placental samples (10 of each fetalsex), and by using HPRT1 as a gene that is expressed significantly differ-ently between samples (lower Cq values in boy placentas, P¼0.017).

Results:Our calculation demonstrates that the RPL30eGAPDH referencegene combination is the better option to assess small placental sex dif-ferences in mRNA level than the reference genes selected define usingGeNorm (YWHAZ and TBP) or NormFinder (TBP and IPO8). The HPRT1normalized mRNA expression level was shown to be different betweenplacental sexes using RPL30 and GAPDH as reference genes (P¼0.01)while not being significantly different using the combination of genessuggested by GeNorm or NormFinder.

Conclusion:This study demonstrates that our new equation is appropriateto assess small variations in mRNA expression between 2 groups such asboy and girl placentas.

Type de document: Document issu d'une conférence ou d'un atelier
Informations complémentaires: Affiche scientifique P1.37 Placenta 57: 260
Mots-clés libres: -
Centre: Centre INRS-Institut Armand Frappier
Date de dépôt: 26 mars 2019 05:01
Dernière modification: 26 mars 2019 05:01
URI: http://espace.inrs.ca/id/eprint/8001

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