Validation of reference genes for gene expression studies in bovine oocytes and cumulus cells derived from in vitro maturation

Abstract

Quantitative real-time PCR (qPCR) is a valuable tool for gene expression studies and it is necessary to choose an ideal endogenous reference gene for data normalization. This work studied a set of reference genes in oocytes and cumulus cells of COCs (Cumulus-Oocyte Complexes) that are suitable for relative gene expression analyses after in vitro maturation (IVM) in bovine. Immature COCs were collected from ovaries of Nelore cattle (Bos indicus) and submitted to IVM. MII oocytes and cumulus cells were subjected to RNA extraction, reverse transcription and preamplification of cDNA. The expression level of eight reference genes (ACTB, GADPH, B2M, H2AFZ, GUSB, HPRT1, PPIA, and TBP) was measured by real time PCR and analyzed by geNorm software. The gene stability measure (M) was calculated and the ideal number of reference genes (RGs) was determined by the V value (pairwise variation). For oocyte samples, two RGs were the ideal number for relative quantification: HPRT1 and B2M and for bovine cumulus samples four were indicated: HPRT1, PPIA, B2M, and TBP genes. The normalization of a non-reference target gene (SOD1) by these reference genes was shown to be considerably different from normalization by less stable reference genes. Our results strengthen the importance of choosing good normalizing genes in order to analyze gene expression under specific experimental conditions and we suggest the use of these RGs in oocytes and cumulus cells of bovine cattle in in vitro matured COCs.

TATA box binding protein and ribosomal protein 4 are suitable reference genes for normalization during quantitative polymerase chain reaction study in bovine mesenchymal stem cells

“…Using other bovine specimens, several studies have been conducted to validate the stability of RGs in each experimental condition. Consistent with the present study results depicted in Figure 3 to 5 , TBP was stably expressed in several bovine tissues, including the cumulus cell [ 16 ], corpus luteum obtained from cyclic or pregnant cows [ 17 ], and liver and thyroid [ 18 ]. RPL15 , a ribosomal protein family along with RPL4 , was also found to show stable expression in oocytes collected from cattle during winter and summer [ 19 ].…”

“…In agreement with the results of the present study, the usage of 18S for normalization was not recommended in the bovine muscular tissue [ 20 ] and corpus luteum [ 17 ]. In addition, several experimental conditions with cattle specimens in terms of the mammary gland under different lactation periods [ 21 ], polymorphonuclear leukocytes [ 22 ], cumulus cell [ 16 ], muscular tissue [ 20 ], and peripheral lymphocytes [ 23 ] were found to be unsuitable to use ACTB and GAPDH for the normalization step. On the other hand, using some conditions such as polymorphonuclear leukocytes [ 22 ] and embryos produced in vitro [ 24 , 25 ] in cattle, 18S and GAPDH were validated as stable RGs, respectively.…”

Human pericentromeric tandemly repeated DNA is transcribed at the end of oocyte maturation and is associated with membraneless mitochondria-associated structures

“…The non-stable transcription of GAPdH during bovine oogenesis especially during the GV-MII period, when its level is tenfold downregulated, www.nature.com/scientificreports/ www.nature.com/scientificreports/ was demonstrated 37 . However, GAPdH is considered to be one of the most stable references for porcine maturating oocytes 38 . Therefore we decided to use both GAPdH and β-actin references.…”

Reverse transcription priming methods affect normalisation choices for gene expression levels in oocytes and early embryos

“…More importantly, geNorm is the most commonly used normalisation algorithm, because geNorm does not need large data sets and the raw data for this program do not need to be normally distributed ( Mehdi Khanlou and Van Bockstaele, 2012 ). Even though the selection of reference genes for normalisation in bovine oocytes has been documented ( Goossens et al , 2005 ; Khan et al , 2016 ; Caetano et al , 2019 ), the effect of different reverse transcription priming strategies to the choice of reference genes has not been addressed in detail. It has been reported that the combination of suitable reference genes includes HPRT1 and B2M in bovine oocyte cDNA samples synthesised using random primers ( Caetano et al , 2019 ), while the ideal reference genes were ACTB and GAPDH in bovine oocyte cDNA samples synthesised using oligo(dT) primers ( Khan et al , 2016 ).…”

“…Even though the selection of reference genes for normalisation in bovine oocytes has been documented ( Goossens et al , 2005 ; Khan et al , 2016 ; Caetano et al , 2019 ), the effect of different reverse transcription priming strategies to the choice of reference genes has not been addressed in detail. It has been reported that the combination of suitable reference genes includes HPRT1 and B2M in bovine oocyte cDNA samples synthesised using random primers ( Caetano et al , 2019 ), while the ideal reference genes were ACTB and GAPDH in bovine oocyte cDNA samples synthesised using oligo(dT) primers ( Khan et al , 2016 ). Consistently, in our study, even though the same original mRNA and system were applied, suitable reference genes for normalisation were different if reverse transcription priming strategies were different.…”