Abstract
BackgroundQuantitative RT-PCR data are commonly normalized using a reference gene. A reference gene is a transcript which expression does not differ in the tissue of interest independent of the experimental condition. The objective of this study was to evaluate the stability of mRNA expression levels of putative reference genes in three different types of equine tissue, endometrial, testicular, and conceptus tissue.FindingsThe expression stability of four (uterine tissue) and six (testicular and conceptus tissue) was assessed using descriptive data analysis and the software programs Normfinder and geNorm. In uterine samples, 18S showed the largest degree of variation in expression while GAPDH, B2M, and ACTB were stably expressed. B2M and GAPDH were identified as the most stably expressed genes in testicular samples, while 18S showed some extent of regulation between samples. Conceptus tissue overall was characterized by very low variability of the transcripts analyzed with GAPDH, YWHZ, and 18S being the most stably expressed genes.ConclusionsIn equine endometrium, GAPDH, B2M, and ACTB transcript levels are equally stable, while 18S is less stably expressed. In testes and associated structures, B2M and GAPDH are the transcripts showing the least amount of variation, while in conceptus tissue GAPDH, YWHZ, and 18S were identified as the most suitable reference genes. Overall, transcripts analyzed in conceptus tissue were characterized by less variation than transcripts analyzed in uterine and testicular tissue.
Highlights
Quantitative RT-PCR data are commonly normalized using a reference gene
In testes and associated structures, B2M and GAPDH are the transcripts showing the least amount of variation, while in conceptus tissue GAPDH, YWHZ, and 18S were identified as the most suitable reference genes
Endometrial samples 18S showed the highest level of mRNA abundance with a mean Cycle threshold (Ct) value of 9.01 ± 0.25, whereas GAPDH, ACTB, and B2M mRNA showed lower expression levels with mean Ct values of 23.47 ± .21, 21.29 ± 0.23, and 20.64 ± 0.24, respectively
Summary
Quantitative RT-PCR data are commonly normalized using a reference gene. A reference gene is a transcript which expression does not differ in the tissue of interest independent of the experimental condition. Real-time reverse transcription PCR (Real-time RT-PCR), referred to as quantitative PCR (RT-qPCR) is a powerful tool to determine quantitative changes in mRNA expression levels and is widely applied in reproductive biology research. This technology provides a means to compare the abundance of a certain transcript of interest in tissue obtained from different biological statuses or to assess the impact of an experimental treatment on the expression of the gene of interest. Normalization procedures are an essential step in the analysis of real-time RT-PCR data to ensure that observed changes in transcript abundance reflect biological variation rather than systematic variation. The objective of this study was to evaluate the stability of mRNA expression levels of putative reference genes in three different types of equine tissue, endometrial, testicular, and conceptus tissue
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