Pregnancy influences the selection of appropriate reference genes in mouse tissue: Determination of appropriate reference genes for quantitative reverse transcription PCR studies in tissues from the female mouse reproductive axis

This study examined the expression stability of a panel of twelve reference genes in tissues from the female mouse reproductive axis and the uterus. Gene expression studies were carried out using reverse transcriptase quantitative polymerase chain reaction (RT-qPCR). cDNA was synthesised from RNA extracted from hypothalami, pituitaries, ovaries and uteri of female mice at ages representing weaning, puberty and adulthood as well as pregnancy (13 ± 1 days post-coitus) (n = a minimum of 3 at each age and at pregnancy). The reference genes examined included 18s, Actb, Atp5b, B2m, Canx, Cyc1, Eif4a2, Gapdh, Rpl13a, Sdha, Ubc and Ywhaz. The RT-qPCR raw data were imported into the qBASE+ software to analyse the expression stability using GeNorm. These data were also subsequently analysed using other software packages (Delta CT, Normfinder, BestKeeper). A comprehensive ranking was conducted considering all stability rankings generated from the different software analyses. B2m and Eif4a2 deviated from the acceptable range for amplification efficiency and therefore were excluded from the further analyses. The stability of the reference genes is influenced by the software used for the analysis with BestKeeper providing markedly different results than the other analyses. GeNorm analysis of tissues taken at different ages but not including pregnant animals, indicated that the expression of the reference genes is tissue specific with the most stable genes being: in the hypothalamus, Canx ...
Source: Gene - Category: Genetics & Stem Cells Authors: Source Type: research