Normalization of qPCR in platelets – YWHAE a potential genericreference gene
The mRNA of human platelets has been extensively studied and it is generally appreciated that platelets contain mRNA transcripts derived from the megakaryocytes, and they have the ability to translate it into proteins. Additionally, platelets contain microRNA (miRNA) that has been shown to potentially regulate the translation of certain proteins. When quantifying gene expression by quantitative real-time polymerase chain reaction (qPCR), a valid normalization method is required and the use of reference genes is a common and robust approach. It is recommended to perform a proper validation of potential reference genes for each individual experimental setup. Previous studies have mainly been performed using commonly used reference genes for nucleated cells, and to our knowledge there are no global evaluations of the stability of transcripts in platelets. Finding a stable transcript would be valuable for inter-study comparisons, and the aim of this study was to identify one or more stable mRNA transcripts suitable as generic reference genes for mRNA gene expression studies in platelets. Platelets were incubated for 24 h and microarray of platelet mRNA revealed that the levels of YWHAE, B2M, ITM2B, H3F3A, PF4V1 remained similar between 0 and 24 h. Further validation of the stability of these genes together with GAPDH, RN18S1, and PPIA, genes frequently used as reference genes in platelet studies, was performed using qPCR after different in vitro conditions. In addition, inter-individual stability of the genes was analyzed in diabetic patients compared with healthy matched controls. Analysis of gene stability by the software RefFinder revealed that YWHAE, PF4V1, and B2M were the most stable genes in platelets from healthy donors. In addition, YWHAE was stable between subjects. Furthermore, the potential influence of miRNA on the selected genes was investigated by knockdown of Dicer1 in the megakaryocytic cell line MEG01. YWHAE, H3F3A, B2M, and GAPDH remained unchanged over time in MEG01 cells indicating that these genes are not regulated by miRNA and hence are more stably expressed. In conclusion, YWHAE is a stable transcript in platelets and we suggest the use of YWHAE as a generic reference gene in mRNA gene expression studies.