Abstract
To obtain reliable results in quantitative PCR (qPCR) reactions, an endogenous control (EC) gene is needed to correct for systematic variations. In this study, a TaqMan low density array was used to quantify the expression levels of microRNA (miRNA) genes in in vivo fertilized, in vitro fertilized, parthenogenetic and somatic cell nuclear transfer blastocysts. The aim was to identify suitable EC genes for the qPCR analysis of miRNAs in porcine blastocysts. The results showed that thirty-six miRNAs were commonly expressed in the four kinds of embryos and the expression levels of eleven miRNAs were similar in the different embryo types (P-value>0.05). These 11 miRNAs were selected as candidate EC genes for further analysis and, of these, miR-16 was identified as the most stable EC gene by the GeNorm (a tool based on a pair-wise comparison model that calculates the internal control genes stability measure and determines the most reliable pair of EC genes) and NormFinder (an excel plug-in that uses an ANOVA-based model to estimate intra- and inter-group variation to indicate the single most stable EC gene) programs. In addition, a cell number normalization method validated miR-16 as a suitable EC gene for use in future qPCR analysis of miRNAs in porcine blastocysts.
Highlights
MicroRNAs are a large family of short noncoding RNAs (~22 nucleotides), and in mammals, more than 400 different miRNAs have been identified, most of which are well conserved in the different species [1]
Concerns have been expressed regarding the use of Ribosomal RNAs (rRNA) in normalization strategies because they are often expressed at much higher levels than the target miRNAs making it very difficult to quantify an rRNA and a rare miRNA transcript in the same RNA dilution [23]
It has been reported that small nuclear RNA (snRNA) and small nucleolar RNAs (snoRNAs) may exhibit tissue-specific and developmental regulation [24] and RNU6B was found to be less stably expressed than the other genes [25]
Summary
MicroRNAs (miRNAs) are a large family of short noncoding RNAs (~22 nucleotides), and in mammals, more than 400 different miRNAs have been identified, most of which are well conserved in the different species [1]. Real-time quantitative PCR (qPCR) is by far the preferred method for miRNA expression quantification studies because of its sensitivity, specificity and wide dynamic range [13]. In qPCR experiment, several variables including amount and quality of RNA, enzymatic efficiencies and differences between tissues or cells in overall transcriptional activity need to be normalized to produce reliable data and incorporating an endogenous control (EC) gene has been found to be an effective method [14]. No. no consensus on suitable EC genes for the quantitative analysis of miRNAs. To identify suitable EC genes for the qPCR analysis of miRNAs in different kinds of porcine blastocysts, the present study was conducted to quantify the expression levels of the miRNAs in in vivo fertilized (IVO), in vitro fertilized (IVF), parthenogenetic (PA) and somatic cell nuclear transfer (SCNT) blastocysts
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