Evaluation of reference genes for RT-qPCR analysis in wild and cultivated Cannabis

RT-qPCR has been widely used for gene expression analysis in recent years. The accuracy of this technique largely depends on the selection of suitable reference genes. In order to facilitate gene expression analysis in wild and cultivated Cannabis, the expression stability of seven candidate reference genes (ACT2, 18S rRNA, GAPDH, UBQ, TUB, PP2A and EF1α) were assessed in leaves samples of different development stages and different organs of both wild and cultivated Cannabis in the present study. Their expression stabilities were evaluated through three software packages (GeNorm, Normfinder and Bestkeeper). Results showed that UBQ and EF1α were the highly ranked genes in different leaves samples, and PP2A was the most stable reference gene in different organs, while GAPDH was the least stable one. And the validation of the reference genes selected was further confirmed by the expression patterns of MDS and OLS.

Expression levels of 7 reference genes in Cannabis, presented as mean value of Ct in all 20 conditions. Ct values are the average of three replicates.