Identification of optimal reference genes for examination of gene expression in different tissues of fetal yaks

Reverse transcription quantitative real-time PCR (RT-qPCR) is widely used to study the relative abundance of mRNA transcripts because of its sensitivity and reliable quantification. However, the reliability of the interpretation of expression data is influenced by several complex factors, including RNA quality, transcription activity, and PCR efficiency, among others. To avoid experimental errors arising from potential variation, the selection of appropriate reference genes to normalize gene expression is essential. In this study, 10 commonly used reference genes – ACTB, B2M, HPRT1, GAPDH, 18SrRNA, 28SrRNA, PPIA, UBE2D2, SDHA, and TBP – were selected as candidate reference genes for six fetal tissues (heart, liver, spleen, lung, kidney, and forehead skin) of yak (Bos grunniens). The transcription stability of the candidate reference genes was evaluated using geNorm, NormFinder, and BestKeeper. The results showed that the combination of TBP and ACTB provided high-quality data for further study. In contrast, the commonly used reference genes 28SrRNA, SDHA, GAPDH, and B2M should not be used for endogenous controls because of their unstable expression in this study. The reference genes that could be used in future gene expression studies in yaks were indentified.

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Selection of reference genes for gene expression studies in chronically hypoxic heart

Journal of Molecular and Cellular Cardiology ◽

10.1016/j.yjmcc.2018.05.075 ◽

2018 ◽

Vol 120 ◽

pp. 22-23

Author(s):

D. Benak ◽

D. Sotakova ◽

J. Neckar ◽

F. Kolar ◽

M. Hlavackova

Keyword(s):

Gene Expression ◽

Reference Genes ◽

Hypoxic Heart ◽

Expression Studies ◽

Gene Expression Studies ◽

Selection Of

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Selection of reference genes for measuring the expression of aiiO in Ochrobactrum quorumnocens A44 using RT-qPCR

Scientific Reports ◽

10.1038/s41598-019-49474-6 ◽

2019 ◽

Vol 9 (1) ◽

Cited By ~ 3

Author(s):

Dorota M. Krzyżanowska ◽

Anna Supernat ◽

Tomasz Maciąg ◽

Marta Matuszewska ◽

Sylwia Jafra

Keyword(s):

Gene Expression ◽

Quorum Sensing ◽

Reference Genes ◽

Significance Threshold ◽

Expression Studies ◽

Reverse Transcription Quantitative Pcr ◽

Gene Expression Studies ◽

The Stability ◽

Selection Of

Abstract Reverse transcription quantitative PCR (RT-qPCR), a method of choice for quantification of gene expression changes, requires stably expressed reference genes for normalization of data. So far, no reference genes were established for the Alphaproteobacteria of the genus Ochrobactrum. Here, we determined reference genes for gene expression studies in O. quorumnocens A44. Strain A44 was cultured under 10 different conditions and the stability of expression of 11 candidate genes was evaluated using geNorm, NormFinder and BestKeeper. Most stably expressed genes were found to be rho, gyrB and rpoD. Our results can facilitate the choice of reference genes in the related Ochrobactrum strains. O. quorumnocens A44 is able to inactivate a broad spectrum of N-acyl homoserine lactones (AHLs) – the quorum sensing molecules of many Gram-negative bacteria. This activity is attributed to AiiO hydrolase, yet it remains unclear whether AHLs are the primary substrate of this enzyme. Using the established RT-qPCR setup, we found that the expression of the aiiO gene upon exposure to two AHLs, C6-HLS and 3OC12-HSL, does not change above the 1-fold significance threshold. The implications of this finding are discussed in the light of the role of quorum sensing-interfering enzymes in the host strains.

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