Abstract
MicroRNA (miRNA) plays essential roles in post-transcriptional regulation of protein coding genes, and the quantitative real-time polymerase chain reaction (qRT-PCR) is the powerful and broadly employed tool to conduct studies of miRNA expression. Identifying appropriate references to normalize quantitative data is a prerequisite to ensure the qRT-PCR accuracy. Until now, there has been no report about miRNA reference for qRT-PCR in Japanese flounder (Paralichthys olivaceus), one important marine cultured fish along the coast of Northern Asia. In this study, combined with miRNA-Seq analysis and literature search, 10 candidates (miR-34a-5p, miR-205-5p, miR-101a-3p, miR-22-3p, miR-23a-3p, miR-210-5p, miR-30c-5p, U6, 5S rRNA, and 18S rRNA) were chosen as potential references to test their expression stability among P. olivaceus tissues, and in livers of P. olivaceus infected with Edwardsiella tarda at different time points. The expression stability of these candidates was analyzed by qRT-PCR and evaluated with Delta CT, BestKeeper, geNorm, as well as NormFinder methods, and RefFinder was employed to estimate the comprehensive ranking according to the four methods. As the result, miR-22-3p and miR-23a-3p were proved to be the suitable combination as reference miRNAs for both P. olivaceus normal tissues and livers infected with E. tarda, and they were successfully applied to normalize miR-7a and miR-221-5p expression in P. olivaceus livers in response to E. tarda infection. All these results provide valuable information for P. olivaceus miRNA quantitative expression analysis in the future.
Highlights
MicroRNA is the small non-coding RNA with a length of about22–24 nucleotide, which exists broadly in diverse organisms and plays important regulatory roles in gene expression at the post-transcription level [1]
Our findings suggest using appropriate reference miRNAs, which will be beneficial in quantitative real-time PCR (qRT-PCR) studies of miRNA expression in Japanese flounder under normal and infectious conditions
The blank control (BC) group was not treated with any solution, while Ringer’s solution (RS) and Edwardsiella tarda-challenged (EC) groups were intraperitoneally injected with 1 mL
Summary
MicroRNA (miRNA) is the small non-coding RNA (ncRNA) with a length of about22–24 nucleotide (nt), which exists broadly in diverse organisms and plays important regulatory roles in gene expression at the post-transcription level [1]. MicroRNA (miRNA) is the small non-coding RNA (ncRNA) with a length of about. To explore the function of miRNA, its expression pattern commonly needs to be verified. There are several methods to quantify miRNA expression, including northern blot, microarray, and quantitative real-time PCR (qRT-PCR) [10,11,12]. QRT-PCR is one of the most widely used technique to quantify miRNA expression due to several advantages, such as good accuracy, high sensitivity, quick reaction, broad application, as well as low cost [13]. Differences in RNA quality and reverse transcription efficiency may lead to inaccuracy in qRT-PCR. It is essential to employ an appropriate reference to normalize the expression of the target RNA molecules [14]
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