Abstract
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 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.
Highlights
Ectopic pregnancy is a common early pregnancy complication in which the embryo implants outside the uterus
We evaluated the effects of using each of these genes on the expression of a test gene of interest in order to demonstrate the effects that arbitrary selection of reference genes may have on data interpretation
The results of our analysis indicated that depending on the reference gene selected, NAPE-PLD mRNA appeared either unchanged in all three study groups (e.g., glyceraldehyde-3-phosphate dehydrogenase (GAPDH), Topoisomerase (DNA) 1 (TOP1)), attenuated in the luteal phase and ectopic pregnancy compared to the follicular phase (e.g., Ubiquitin C (UBC), YWHAZ) or that expression may be increased in the luteal phase compared to the follicular phase or ectopic pregnancy (e.g., RPL13A) (Figure 5)
Summary
Ectopic pregnancy is a common early pregnancy complication in which the embryo implants outside the uterus. RT-qPCR is an important and common technique used to examine changes in gene expression levels in various pathological states It offers an advantage over other methods such as northern blotting because of its high sensitivity, specificity, ease of use and broad dynamic range [5,6]. This feature albeit advantageous comes with a price i.e., that it is susceptible to variation in extraction and experimental processes including differences in RNA extraction, presence of contaminating DNA, efficiency of cDNA synthesis etc. One of the most common methods of normalisation is the use of a reference gene whose expression should remain constant under various experimental and disease conditions [10]. The validity of gene expression results generated by this method of normalisation is highly dependent on the optimal selection of endogenous reference genes to which the genes of interest are to be normalised
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