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.
Highlights
MicroRNAs are a recently discovered class of bioregulatory, short, non-coding molecules that bind to target messenger RNAs and repress their translation
To minimize experimental errors which may occur at any step of the RNA to cDNA to PCR transition or between PCR runs, relative quantification employs a reference gene to normalize the measurement of transcript levels in experimental samples
This study aims to determine the suitability of six potential reference genes for miRNA quantification by real time PCR during early gestation in the pig
Summary
MicroRNAs (miRNAs) are a recently discovered class of bioregulatory, short, non-coding molecules that bind to target messenger RNAs (mRNAs) and repress their translation. This is achieved by physically inhibiting translation of the mRNA or through its degradation [1,2]. Real time PCR is a sensitive method of measuring gene transcript levels in biological systems. It has recently become a popular method of measuring miRNA expression [9,10,11,12,13,14,15,16,17,18]. As miRNA gene expression profiling is an emerging methodology, few reports have been published which identify suitable reference genes for real time PCR studies
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