Identification and validation of suitable endogenous reference genes for gene expression studies in human peripheral blood

Oat (Avena sativa L.) is a widely cultivated cereal with high nutritional value and it is grown mainly in temperate regions. The number of studies dealing with gene expression changes in oat continues to increase, and to obtain reliable RT-qPCR results it is essential to establish and use reference genes with the least possible influence caused by experimental conditions. However, no detailed study has been conducted on reference genes in different tissues of oat under diverse abiotic stress conditions. In our work, nine candidate reference genes (ACT, TUB, CYP, GAPD, UBC, EF1, TBP, ADPR, PGD) were chosen and analysed by four statistical methods (GeNorm, Normfinder, BestKeeper, RefFinder). Samples were taken from two tissues (leaves and roots) of 13-day-old oat plants exposed to five abiotic stresses (drought, salt, heavy metal, low and high temperatures). ADPR was the top-rated reference gene for all samples, while different genes proved to be the most stable depending on tissue type and treatment combinations. TUB and EF1 were most affected by the treatments in general. Validation of reference genes was carried out by PAL expression analysis, which further confirmed their reliability. These results can contribute to reliable gene expression studies for future research in cultivated oat.

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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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