Gene expression analysis of pig cumulus-oocyte complexes stimulated in vitro with follicle stimulating hormone or epidermal growth factor-like peptides

Blaha, Milan; Němcová, Lucie; Kepková, Kateřina Vodičková; Vodička, Petr; Procházka, Radek

doi:10.1186/s12958-015-0112-2

Cited by 43 publications

(33 citation statements)

References 97 publications

(113 reference statements)

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…Widely used reference genes for expression analyses include GAPDH , ACTB , and HPRT (Blaha et al, 2015; Boosani, Dhar & Agrawal, 2015; Wang et al, 2016; Zhang et al, 2016b; Zhao et al, 2015). These genes are reported to have consistent expression levels under various conditions such as different organs and different developmental stages (Tang et al, 2007).…”

Section: Introductionmentioning

confidence: 99%

Identifying suitable reference genes for gene expression analysis in developing skeletal muscle in pigs

Niu

Yang

Zhang

et al. 2016

PeerJ

View full text Add to dashboard Cite

The selection of suitable reference genes is crucial to accurately evaluate and normalize the relative expression level of target genes for gene function analysis. However, commonly used reference genes have variable expression levels in developing skeletal muscle. There are few reports that systematically evaluate the expression stability of reference genes across prenatal and postnatal developing skeletal muscle in mammals. Here, we used quantitative PCR to examine the expression levels of 15 candidate reference genes (ACTB, GAPDH, RNF7, RHOA, RPS18, RPL32, PPIA, H3F3, API5, B2M, AP1S1, DRAP1, TBP, WSB, and VAPB) in porcine skeletal muscle at 26 different developmental stages (15 prenatal and 11 postnatal periods). We evaluated gene expression stability using the computer algorithms geNorm, NormFinder, and BestKeeper. Our results indicated that GAPDH and ACTB had the greatest variability among the candidate genes across prenatal and postnatal stages of skeletal muscle development. RPS18, API5, and VAPB had stable expression levels in prenatal stages, whereas API5, RPS18, RPL32, and H3F3 had stable expression levels in postnatal stages. API5 and H3F3 expression levels had the greatest stability in all tested prenatal and postnatal stages, and were the most appropriate reference genes for gene expression normalization in developing skeletal muscle. Our data provide valuable information for gene expression analysis during different stages of skeletal muscle development in mammals. This information can provide a valuable guide for the analysis of human diseases.

show abstract

Section: Introductionmentioning

confidence: 99%

Identifying suitable reference genes for gene expression analysis in developing skeletal muscle in pigs

Niu

Yang

Zhang

et al. 2016

PeerJ

View full text Add to dashboard Cite

show abstract

“…In pig cumulus cells, we found also that the most stable reference genes ( GAPDH/18S ) and expression dynamics of each reference gene during IVM were different from that of pig oocytes. In addition, although mRNA abundance studies in pig cumulus cells have been conducted frequently using HPRT/BACT/GAPDH as single internal control (Blaha, Nemcova, Kepkova, Vodicka, & Prochazka, ; Blaha, Nemcova, & Prochazka, and Yang et al., ), the systematic study on the selection of reference genes has not been performed yet until now. Thus, the combination of GAPDH/18S suitable as references for pig cumulus cells found in the present study could be used in future investigations.…”

Section: Discussionmentioning

confidence: 99%

Methods of RNA preparation affect mRNA abundance quantification of reference genes in pig maturing oocytes

2017

Reprod Domestic Animals

View full text Add to dashboard Cite

To ensure accurate normalization and quantification of target RNA transcripts using reverse transcription quantitative polymerase chain reaction (RT-qPCR), most studies focus on the identification of stably expressed gene(s) as internal reference. However, RNA preparation methods could also be an important factor, especially for test samples of limited quantity (e.g. oocytes). In this study, we aimed to select appropriate reference gene(s), and evaluate the effect of RNA preparation methods on gene expression quantification in porcine oocytes and cumulus cells during in vitro maturation. Expression profiles of seven genes (GAPDH, 18S, YWHAG, BACT, RPL4, HPRT1 and PPIA) were examined, on RNA samples extracted from cumulus cells (RNeasy Kit) and oocytes (RNeasy Kit and Lysis Kit) during in vitro maturation, respectively. Interestingly, different RNA preparation methods were found to potentially affect the quantification of reference gene expression in pig oocytes cultured in vitro. After geNorm analyses, the most suitable genes for normalization were identified, GAPDH/18S for cumulus cells and YWHAG/BACT for oocytes, respectively. Thus, our results provide useful data and information on the selection of better reference genes and RNA preparation method for related functional studies.

show abstract

“…Blaha et alstudied the effect of FSH on ID1 gene expression pattern in porcine cumulus cells with microarray assays. They found that FSH administration led to increased expression of ID1 gene, and concluded that, since ID1 is responsible for cumulus cells differentiation and processes leading to successful IVM, may be potentially marker gene of oocyte maturational competence in vitro [2]. Hogg et al presented evidence that ID1 acts during embryogenesis and development promoting cell growth in sheep.…”

Section: Discussionmentioning

confidence: 99%

Does Porcine Oocytes Maturation in Vitro is Regulated by Genes Involved in Transforming Growth Factor Beta Receptor Signaling Pathway?

Budna

Celichowski

Karimi

et al. 2017

Advances in Cell Biology

View full text Add to dashboard Cite

Summary:The oocyte growth and development in follicular environment are substantially accompanied by surrounding somatic cumulus (CCs) and granulosa cells (GCs). During these processes, the mammalian gametes reach full maturational stage and may be further successfully fertilized by single spermatozoon. These unique mechanisms are regulated by expression of clusters of genes and their biochemical signaling pathways. In this article we described differential expression pattern of transforming growth factor beta (TGFB) gene superfamily in porcine oocytes before and after in vitro maturation (IVM). We performed Affymetrix® microarray assays to investigate the TGFB-related genes expression profile in porcine immature oocytes and gametes cultured for 44h in vitro. In results we found 419 different genes, 379 genes with lower expression, and 40 genes characterized by increased RNA profile. Moreover, significant up-regulation of 6 genes belonging to TGFB signaling pathway such as: TGFBR3, SMAD4, FOS, KLF10, ID1, MAP3K1 in immature porcine oocytes (before IVM), was also observed. It may be suggested that genes involved in TGFB-related signaling pathway are substantially regulated before IVM. Furthermore, these genes may play a significant role during early stages of nuclear and/or cytoplasmic porcine oocytes maturation. The investigated transcripts may be also recommended as the markers of oocytes maturational capability in pigs.

show abstract

Gene expression analysis of pig cumulus-oocyte complexes stimulated in vitro with follicle stimulating hormone or epidermal growth factor-like peptides

Cited by 43 publications

References 97 publications

Identifying suitable reference genes for gene expression analysis in developing skeletal muscle in pigs

Identifying suitable reference genes for gene expression analysis in developing skeletal muscle in pigs

Methods of RNA preparation affect mRNA abundance quantification of reference genes in pig maturing oocytes

Does Porcine Oocytes Maturation in Vitro is Regulated by Genes Involved in Transforming Growth Factor Beta Receptor Signaling Pathway?

Contact Info

Product

Resources

About