RNA extraction and expression analysis by quantitative PCR (qPCR)

LL L. M. Legault
KD K. Doiron
MB M. Breton-Larrivée
AL A. Langford-Avelar
AL A. Lemieux
MC M. Caron
LJ L. A. Jerome-Majewska
DS D. Sinnett
SM S. McGraw
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Quantitative gene expression analyses were performed as previously [122, 123]. Briefly, embryonic E10.5 forebrains were isolated, flash frozen and kept at − 80 °C until RNA extraction. RNA was extracted using RNeasy Mini kit (Qiagen #74004) following manufacturer’s recommendations. Extracted RNA was quantified using QuBit fluorimeter apparel with the High Sensitivity RNA assay kit (ThermoFisher #Q32852). 600 ng of RNA was used for cDNA conversion using SuperScript IV Reverse Transcriptase (ThermoFisher #18090010). For each gene (primer sequence Additional file 1: Table S2), qPCR reactions were performed in triplicate on 5 ng of cDNA using SensiFAST SYBR No-ROX (Bioline #BIO-98005) on a LightCycler 96 (Roche Life Science). Gene expression analysis and normalization was done using the 2−∆∆Ct method using Hprt1 and Pgk1 as reference genes. Statistical analyses were done using GraphPad prism (version 8.4.3) for t test with Welch correction.

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