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Variation in stability of endogenous reference genes in fallopian tubes and endometrium from healthy and ectopic pregnant women.

ABSTRACT: RT-qPCR is commonly employed in gene expression studies in ectopic pregnancy. Most use RN18S1, ?-actin or GAPDH as internal controls without validation of their suitability as reference genes. A systematic study of the suitability of endogenous reference genes for gene expression studies in ectopic pregnancy is lacking. The aims of this study were therefore to evaluate the stability of 12 reference genes and suggest those that are stable for use as internal control genes in fallopian tubes and endometrium from ectopic pregnancy and healthy non-pregnant controls. Analysis of the results showed that the genes consistently ranked in the top six by geNorm and NormFinder algorithms, were UBC, GAPDH, CYC1 and EIF4A2 (fallopian tubes) and UBC and ATP5B (endometrium). mRNA expression of NAPE-PLD as a test gene of interest varied between the groups depending on which of the 12 reference genes was used as internal controls. This study demonstrates that arbitrary selection of reference genes for normalisation in RT-qPCR studies in ectopic pregnancy without validation, risk producing inaccurate data and should therefore be discouraged.

SUBMITTER: Gebeh AK

PROVIDER: S-EPMC3317689 | biostudies-literature | 2012

REPOSITORIES: biostudies-literature

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Variation in stability of endogenous reference genes in fallopian tubes and endometrium from healthy and ectopic pregnant women.

Gebeh Alpha K AK Marczylo Emma L EL Amoako Akwasi A AA Willets Jonathon M JM Konje Justin C JC

International journal of molecular sciences 20120302 3

RT-qPCR is commonly employed in gene expression studies in ectopic pregnancy. Most use RN18S1, β-actin or GAPDH as internal controls without validation of their suitability as reference genes. A systematic study of the suitability of endogenous reference genes for gene expression studies in ectopic pregnancy is lacking. The aims of this study were therefore to evaluate the stability of 12 reference genes and suggest those that are stable for use as internal control genes in fallopian tubes and e ...[more]

PMID: 22489127

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Project description:Cytomegalovirus (CMV) has recently been suggested as a potential risk factor for the development of ectopic pregnancy (EP) following upper genital tract infection in women. However, little is known about its associated underlying pathogenic mechanisms. This was a prospective case-control study that measured the prevalence of CMV infection in Fallopian tubes (FT) bearing an EP and its effects on the tubal expression of interleukin (IL)-6 and its signaling molecules, which are known to play significant roles in the immune response against CMV infection as well as embryo implantation. Fresh FTs from 96 EPs during salpingectomy and another 61 women at the midluteal phase during total abdominal hysterectomy (TAH) were collected to measure the rate of CMV by an IVD CE PCR kit. The participants were then classified to measure the expression of IL-6, its receptor (IL6R) and intracellular mediators (gp-130, STAT3) by immunohistochemistry and quantitative RT-PCR. The results showed significantly higher (P = 0.01) rates of CMV in FTs obtained from EP (22.9 %) compared with controls (8.2 %). IL-6 (P = 0.003), IL6R? (P = 0.02), gp 130 (P = 0.008) and STAT3 (P = 0.03) were significantly higher in TAH-positive (n = 5) compared with TAH-negative FTs by immunohistochemistry. Furthermore, the expression in the non-infected EP samples was significantly higher for IL-6 (P = 0.004), IL6R (P = 0.007), gp130 (P = 0.006) and STAT3 (P = 0.007) compared with negative TAH. Similar results were observed by quantitative PCR. CMV-positive EP samples showed the highest significant increase of the studied molecules by all techniques. In conclusion, Fallopian tubal infection with CMV is higher in EP and could predispose to embryo implantation by up-regulating the expression of IL-6 and its related molecules as part of tubal innate immune response. Further in vitro and in vivo studies are compulsory to illustrate the roles of IL-6 and CMV in the pathogenesis of EP.

Dataset Information

Variation in stability of endogenous reference genes in fallopian tubes and endometrium from healthy and ectopic pregnant women.

Publications

Variation in stability of endogenous reference genes in fallopian tubes and endometrium from healthy and ectopic pregnant women.

Similar Datasets

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