The Selection of Reference Genes for Quantitative Real-Time PCR in the Ashidan Yak Mammary Gland During Lactation and Dry Period

Investigating the critical genes related to milk synthesis is essential for the improvement of the milk yield of the yak. Real-time quantitative polymerase chain reaction (RT-qPCR) is a reliable and widely used method to measure and evaluate gene expression levels. Selection of suitable reference genes is mandatory to acquire accurate normalization of gene expression results from RT-qPCR. To select the most stable reference genes for reliable normalization of mRNA expression by RT-qPCR in the mammary gland of the Ashidan yak, we selected 16 candidate reference genes and analyzed their expression stability at different physiological stages (lactation and dry period). The expression stability of the candidate reference genes was assessed using geNorm, NormFinder, BestKeeper, Delta Ct, and RefFinder methods. The results showed that the hydroxymethylbilane synthase gene (HMBS) and the tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, zeta polypeptide gene (YWHAZ) were the most stable genes across all treatment samples. The reliability of selected reference genes was validated by normalizing relative expression of the lactation-related 60S ribosomal protein L35 gene (RPL35). The relative expression of RPL35 varied considerably according to the different reference genes. This work provides valuable information to further promote research in the molecular mechanisms involved in lactation and mammary gland development and provides a foundation for the improvement of the milk yield and quality of the Ashidan yak.

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Identification of reliable reference genes for gene expression studies in maternal reproductive tissues and fetal tissues of pregnant cows

10.21203/rs.2.22639/v1 ◽

2020 ◽

Author(s):

Lei Cheng ◽

Jie Yu ◽

Xiuzhong Hu ◽

Min Xiang ◽

Yu Xia ◽

...

Keyword(s):

Gene Expression ◽

Reference Genes ◽

Target Genes ◽

Quantitative Polymerase Chain Reaction ◽

Related Factor ◽

Expression Stability ◽

Relative Expression ◽

Reproductive Tissues ◽

Fetal Tissues ◽

Selection Of

Abstract Background: The relationship between the conceptus and the maternal uterine environment is crucial for the successful establishment and maintenance of pregnancy in cattle. Gene expression analysis of the conceptus and maternal reproductive tissues is a favorable method to assess the embryonic maternal interaction. The reliability of the commonly used method reverse transcription-quantitative polymerase chain reaction (RT-qPCR) depends on proper normalization to stable reference genes (RGs). The objective of this study was to determine the expression stability of ten potential RGs (SUZ12, CNOT11, ACTB, RPL19, RPS9, GAPDH, TBP, HPRT1, SDHA and PPIA) in maternal reproductive tissues and fetal tissues, and to analyze the effect of RG selection on the calculation of the relative expression of target genes. Results: The expression stability of ten potential RGs was analyzed in eight different tissues (caruncular endometrium, intercaruncular endometrium, corpus luteum, ovary, oviduct, mammary gland, embryonic disc and trophoblast) from three pregnant dairy cows. Three programs—GeNorm, NormFinder and Bestkeeper—were used to identify the best RGs. According to all three programs, the most stable RG was CNOT11, whereas the least stable RGs were GAPDH and HPRT1. GeNorm analysis showed that a combination of five RGs (SDHA, PPIA, CNOT11, RPS9 and RPL19) was necessary for appropriate data normalization. However, NormFinder analysis indicated that the combination of CNOT11 and PPIA was the most suitable. When target genes were normalized to these RGs, the relative expression of the Radical S-adenosyl methionine domain containing 2 gene was not affected by the choice of RGs, whereas a large difference was observed in the expression profile of the Nuclear erythroid2-related factor 2 gene between the most stable RGs and least stable RGs. Conclusions: The results indicate that careful selection of RGs is crucial under different conditions, especially for target genes with relatively small fold changes. Furthermore, the results provide useful information for the selection of RGs for evaluating genes affecting bovine reproduction.

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Cloning and evaluation of reference genes for quantitative real-time PCR analysis inAmorphophallus

PeerJ ◽

10.7717/peerj.3260 ◽

2017 ◽

Vol 5 ◽

pp. e3260 ◽

Cited By ~ 4

Author(s):

Kai Wang ◽

Yi Niu ◽

Qijun Wang ◽

Haili Liu ◽

Yi Jin ◽

...

Keyword(s):

Gene Expression ◽

Heat Stress ◽

Real Time ◽

Reference Genes ◽

Dynamic Range ◽

Expression Profiles ◽

Mrna Levels ◽

Expression Stability ◽

Degenerate Primers ◽

Different Tissues

Quantitative real-time reverse transcription PCR (RT-qPCR) has been widely used in the detection and quantification of gene expression levels because of its high accuracy, sensitivity, and reproducibility as well as its large dynamic range. However, the reliability and accuracy of RT-qPCR depends on accurate transcript normalization using stably expressed reference genes.Amorphophallusis a perennial plant with a high content of konjac glucomannan (KGM) in its corm. This crop has been used as a food source and as a traditional medicine for thousands of years. Without adequate knowledge of gene expression profiles, there has been no report of validated reference genes inAmorphophallus. In this study, nine genes that are usually used as reference genes in other crops were selected as candidate reference genes. These putative sequences of these genesAmorphophalluswere cloned by the use of degenerate primers. The expression stability of each gene was assessed in different tissues and under two abiotic stresses (heat and waterlogging) inA. albusandA. konjac. Three distinct algorithms were used to evaluate the expression stability of the candidate reference genes. The results demonstrated thatEF1-a,EIF4A,H3andUBQwere the best reference genes under heat stress inAmorphophallus. Furthermore,EF1-a,EIF4A,TUB, andRPwere the best reference genes in waterlogged conditions. By comparing different tissues from all samples, we determined thatEF1-α,EIF4A,andCYPwere stable in these sets. In addition, the suitability of these reference genes was confirmed by validating the expression of a gene encoding the small heat shock proteinSHSP, which is related to heat stress inAmorphophallus. In sum,EF1-αandEIF4Awere the two best reference genes for normalizing mRNA levels in different tissues and under various stress treatments, and we suggest using one of these genes in combination with 1 or 2 reference genes associated with different biological processes to normalize gene expression. Our results will provide researchers with appropriate reference genes for further gene expression quantification using RT-qPCR inAmorphophallus.

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Selection of reference genes for quantitative real-time PCR normalisation in adipose tissue, muscle, liver and mammary gland from ruminants

animal ◽

10.1017/s1751731113000475 ◽

2013 ◽

Vol 7 (8) ◽

pp. 1344-1353 ◽

Cited By ~ 64

Author(s):

M. Bonnet ◽

L. Bernard ◽

S. Bes ◽

C. Leroux

Keyword(s):

Adipose Tissue ◽

Mammary Gland ◽

Real Time ◽

Real Time Pcr ◽

Reference Genes ◽

Quantitative Real Time Pcr ◽

Selection Of

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Expression stability and selection of optimal reference genes for gene expression normalization in early life stage rainbow trout exposed to cadmium and copper

Aquatic Toxicology ◽

10.1016/j.aquatox.2017.07.009 ◽

2017 ◽

Vol 190 ◽

pp. 217-227 ◽

Cited By ~ 11

Author(s):

Kamran Shekh ◽

Song Tang ◽

Som Niyogi ◽

Markus Hecker

Keyword(s):

Gene Expression ◽

Rainbow Trout ◽

Reference Genes ◽

Early Life ◽

Life Stage ◽

Early Life Stage ◽

Expression Stability ◽

Selection Of

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Selection of appropriate reference genes for the detection of rhythmic gene expression via quantitative real-time PCR in Tibetan hulless barley

PLoS ONE ◽

10.1371/journal.pone.0190559 ◽

2018 ◽

Vol 13 (1) ◽

pp. e0190559 ◽

Cited By ~ 9

Author(s):

Jing Cai ◽

Pengfei Li ◽

Xiao Luo ◽

Tianliang Chang ◽

Jiaxing Li ◽

...

Keyword(s):

Gene Expression ◽

Real Time ◽

Real Time Pcr ◽

Reference Genes ◽

Quantitative Real Time Pcr ◽

Hulless Barley ◽

Rhythmic Gene ◽

Tibetan Hulless Barley ◽

Selection Of

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Selection of suitable reference genes for abiotic stress-responsive gene expression studies in peanut by real-time quantitative PCR

Electronic Journal of Biotechnology ◽

10.1016/j.ejbt.2017.05.004 ◽

2017 ◽

Vol 28 ◽

pp. 76-86 ◽

Cited By ~ 6

Author(s):

Meijing He ◽

Shunli Cui ◽

Xinlei Yang ◽

Guojun Mu ◽

Huanying Chen ◽

...

Keyword(s):

Gene Expression ◽

Abiotic Stress ◽

Real Time ◽

Quantitative Pcr ◽

Reference Genes ◽

Real Time Quantitative Pcr ◽

Expression Studies ◽

Gene Expression Studies ◽

Suitable Reference ◽

Selection Of

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Systematic selection and validation of suitable reference genes for quantitative real-time PCR normalization studies of gene expression in Nitraria tangutorum

Scientific Reports ◽

10.1038/s41598-020-73059-3 ◽

2020 ◽

Vol 10 (1) ◽

Author(s):

Bo Wang ◽

Huirong Duan ◽

Peifang Chong ◽

Shiping Su ◽

Lishan Shan ◽

...

Keyword(s):

Gene Expression ◽

Real Time ◽

Real Time Pcr ◽

Reference Genes ◽

Expression Stability ◽

Quantitative Real Time Pcr ◽

Experimental Conditions ◽

Nitraria Tangutorum ◽

Multiple Species ◽

Suitable Reference

Abstract Suitable reference genes can be used to calibrate the error in quantitative real-time PCR (qPCR) experiments, making the results more credible. However, there are no reference genes suitable for multiple species and under different experimental conditions. Nitraria tangutorum Bobr. is a typical plant native to desert areas. It is drought-resistant, saline-alkali resistant, extreme temperatures-resistant, and has strong adaptability. To date, the importance of this germplasm has not been sufficiently understood; therefore, it is still unclear which genes can be used as reference genes to calibrate qPCR data of N. tangutorum. In this study we analyzed the expression levels of 10 candidate reference genes (ACT, GAPDH, TUA, TUB, CYP, UBC, His, PP2A, HSP, and EF1-α) in N. tangutorum seedlings under a series of experimental conditions, including in different organs (root, stem, and leaf) and under abiotic stresses (salt, drought, heat, and cold) and hormone stimuli (abscisic acid) by qPCR. Three software programs (geNorm, NormFinder, and BestKeeper) were used to evaluate the expression stability of the ten genes. Comprehensive analysis showed that EF1-α and His had the best expression stability, whereas HSP was the least suitable as a reference gene. The expression profile of NtCER7, a gene related to the regulation of cuticular wax biosynthesis in N. tangutorum, verified the accuracy of the experimental results. Based on this study, we recommend EF1-α and His as suitable reference genes for N. tangutorum. This paper provides the first data on stable reference genes in N. tangutorum, which will be beneficial to studying the gene expression of N. tangutorum and other Nitraria species in the future.

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