Development of Plasmid Calibrators for Absolute Quantification of miRNAs by Using Real-Time qPCR

Abstract

MicroRNAs (miRNAs) are small noncoding RNAs of approximately 18 to 25 nucleotides in length that negatively regulate gene expression via either the degradation or translational inhibition of their target mRNAs. Because miRNAs are essential for the regulation of critical physiological processes as well as a variety of pathological events, they have emerged as a novel class of molecular diagnostic biomarkers and therapeutic agents or targets. Accordingly, the need for novel methods for the quantification of miRNA has increased due to interest in their clinical implications. Currently, real-time quantitative polymerase chain reaction (qPCR) is considered the most robust technology for nucleic acid quantification. Different tools for miRNA quantification by using qPCR are now commercially available, but only relative quantification strategies have been reported. This situation may be partly due to the difficulty in obtaining an appropriate molecule with which to establish an miRNA calibration range. Here, we describe a rapid and convenient strategy for the development of a calibrator, which enables the absolute quantification of miRNAs by using qPCR and allows the cloning of a synthetic sequence of interest instead of a PCR product into a plasmid.

Differential expression of plasma miR-146a in sepsis patients compared with non-sepsis-SIRS patients

“…There are several types of calibrators for absolute quantification of miRNAs by qRT-PCR, including plasmid (20), RNA fraction (21), cDNA samples (22) and synthetic target miRNA (23). For relative qRT-PCR, an appropriate reference gene must be selected.…”

Absolute quantification of microRNAs in green tea (Camellia sinensis) by stem‐loop quantitative real‐time PCR

“…In recent years, some methods of detection and absolute quantification of miRNAs, including a universal references, plasmid calibrator and DNA–gold nanoparticle probe by means of miRNA microarray, nanotechnological approach and qRT‐PCR have been reported . Among these, stem‐loop qRT‐PCR is currently the most accurate and widely applied method for detection and quantification of small RNAs in plants and in the diet .…”

“…Therefore, it is important to experimentally determine the presence and level of dietary miRNA in a particular system as well as to determine their functional characteristics with regard to improvement of human health . Recently, the detection and absolute quantification of miRNAs through miRNA microarray, qRT‐PCR and nano‐technologies have been reported . Among the techniques studied, conventional techniques, especially qRT‐PCR, were found to be excellent in terms of real‐time efficiency and specificity .…”

MEG3-4 is a miRNA decoy that regulates IL-1β abundance to initiate and then limit inflammation to prevent sepsis during lung infection

“…Absolute copy numbers of mRNA, lncRNA, and miRNA were measured as previously described (52). Plasmid pWT-MEG3 was used for constructing MEG3–4 standard curve, whereas pGL3–IL-1β was used for IL-1β mRNA and pMD19-T–miR-138 for miR-138.…”