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Detection of microRNA Expression in Peritoneal Membrane of Rats Using Quantitative Real-time PCR
Published on: June 27, 2017
Selection and validation of reference genes for miRNA expression studies during porcine pregnancy
Jocelyn M Wessels1, Andrew K Edwards, Candace Zettler
1Department of Biomedical Sciences, Ontario Veterinary College, University of Guelph, Guelph, Canada.
Abstract:
MicroRNAs comprise a family of small non-coding RNAs that modulate several developmental and physiological processes including pregnancy. Their ubiquitous presence is confirmed in mammals, worms, flies and plants. Although rapid advances have been made in microRNA research, information on stable reference genes for validation of microRNA expression is still lacking. Real time PCR is a widely used tool to quantify gene transcripts. An appropriate reference gene must be chosen to minimize experimental error in this system. A small difference in miRNA levels between experimental samples can be biologically meaningful as these entities can affect multiple targets in a pathway. This study examined the suitability of six commercially available reference genes (RNU1A, RNU5A, RNU6B, SNORD25, SCARNA17, and SNORA73A) in maternal-fetal tissues from healthy and spontaneously arresting/dying conceptuses from sows were separately analyzed at gestation day 20. Comparisons were also made with non-pregnant endometrial tissues from sows. Spontaneous fetal loss is a prime concern to the commercial pork industry. Our laboratory has previously identified deficits in vasculature development at maternal-fetal interface as one of the major participating causes of fetal loss. Using this well-established model, we have extended our studies to identify suitable microRNA reference genes. A methodical approach to assessing suitability was adopted using standard curve and melting curve analysis, PCR product sequencing, real time PCR expression in a panel of gestational tissues, and geNorm and NormFinder analysis. Our quantitative real time PCR analysis confirmed expression of all 6 reference genes in maternal and fetal tissues. All genes were uniformly expressed in tissues from healthy and spontaneously arresting conceptus attachment sites. Comparisons between tissue types (maternal/fetal/non-pregnant) revealed significant differences for RNU5A, RNU6B, SCARNA17, and SNORA73A expression. Based on our methodical assessment of all 6 reference genes, results suggest that RNU1A is the most stable reference gene for porcine pregnancy studies.
Insights
Researchers identified RNU1A as the most stable reference gene for microRNA expression analysis in porcine pregnancy. This finding is crucial for accurate studies of fetal loss and reproductive health in pigs.
Area of Science:
- Reproductive Biology
- Molecular Biology
- Genomics
Background:
- MicroRNAs regulate key developmental and physiological processes, including pregnancy.
- Accurate quantification of microRNA expression is essential for understanding biological pathways, but lacks validated reference genes.
- Spontaneous fetal loss in swine impacts the pork industry, with vascular deficits identified as a contributing factor.
Purpose of the Study:
- To identify stable reference genes for microRNA expression analysis in porcine maternal-fetal tissues.
- To compare reference gene stability in healthy, arresting, and non-pregnant porcine tissues.
- To provide a validated reference gene for future studies on porcine pregnancy and fetal loss.
Main Methods:
- Quantitative real-time PCR (qRT-PCR) was used to analyze the expression of six candidate reference genes (RNU1A, RNU5A, RNU6B, SNORD25, SCARNA17, SNORA73A).
- Expression levels were assessed in maternal, fetal, and non-pregnant endometrial tissues from sows at gestation day 20.
- Stability analysis was performed using standard curve, melting curve analysis, PCR product sequencing, geNorm, and NormFinder.
Main Results:
- All six candidate reference genes were expressed in maternal and fetal porcine tissues.
- Uniform expression was observed across healthy and spontaneously arresting conceptus attachment sites.
- Significant differences in expression were found for RNU5A, RNU6B, SCARNA17, and SNORA73A between different tissue types.
Conclusions:
- RNU1A demonstrated the highest stability across maternal-fetal tissues during porcine pregnancy.
- RNU5A, RNU6B, SCARNA17, and SNORA73A showed significant variations, making them less suitable as reference genes.
- RNU1A is recommended as the optimal reference gene for microRNA expression studies in porcine pregnancy and fetal loss research.
