Dye‐Free MicroRNA Quantification by Using Pyrosequencing with a Sequence‐Tagged Stem–loop RT Primer

MicroRNAs (miRNAs) are a class of endogenous, ~22-nucleotide (nt) noncoding RNAs that play an important role in the control of the developmental processes of cells by negative regulation of protein-coding gene expression. To date, there are 17 341 mature miRNAs, including 1048 human miRNAs, in the University of Manchester miRNA database (http://www. mirbase.org/). Although miRNAs represent a relatively abundant class of transcripts, their expression levels vary greatly in different tissue types and species. Analyzing miRNA expression levels in tissues or cells can supply valuable information for investigating the biological functions of miRNAs; however conventional techniques to amplify miRNAs for detection and quantification present a significant challenge because of the short length of these molecules; thus, a number of straightforward methods without the use of amplification have been developed for miRNA detection. Northern blotting 5] is the widely used standard method for analyzing miRNAs; however, relatively large amounts of starting material (RNA) are required for an assay. To improve the sensitivity of miRNA quantification, a method based on splinted ligation was developed. This exhibits approximately 50 times greater sensitivity than Northern blotting, but radioactive P labels are needed. A single-molecule method, based on the hybridization of two spectrally distinguishable LNA–DNA oligonucleotide probes (for the miRNA of interest), offers a direct miRNA assay as sensitive as 500 fm, but an expensive single-molecule detection instrument is required. For sensitive miRNA detection, amplification techniques are thus necessary. By skillfully designing detection probes, a modified “Invader” assay was developed for the quantification of miRNAs. Although 20 000 miRNAs were detected, accurate quantification of miRNAs among samples is difficult because the initial target concentration is proportional to the steady-state reaction rate of “invasive” amplification. In contrast, an miRNA assay based on real-time quantitative PCR with a stem–loop reverse transcription (RT) primer was much more quantitative, as the Ct (cycle threshold) value is inversely proportional to the amount of initial target. However, PCRs of the sample and reference targets are performed separately, and a small difference in amplification efficiency between the sample and the reference yields a large difference in the amount of final product ; this results in large inter-PCR variations. Recently, a simple and sensitive miRNA quantification method that used branched rolling-circle amplification (BRCA) was reported, but quantification based on endpoint readout seems challengeable because of the time-dependent amplification efficiency of BRCA. To achieve accurate quantification of a target miRNA in a sample, real-time monitoring of signal intensities from both a sample and a reference (quantification standard) is necessary, because the reaction rate slows down as the reaction proceeds. As the real-time detection requires a sophisticated instrument, quantification using endpoint data is preferable. In the present study, we have developed a pyrosequencing-based method for absolute quantification, and for comparing the relative miRNA expression levels in biological samples. Pyrosequencing is a well-developed technology for DNA sequencing. It uses cascade enzymatic reactions to monitor the release of inorganic pyrophosphate that results from dNTP incorporation. Because of its highly quantitative performance, pyrosequencing has been widely used for genotyping, and the analysis of DNA methylation and gene expression. Here we employed pyrosequencing technology to quantify microRNAs by quantitatively detecting sequence labels that were artificially tagged into the RT products of miRNA. Unlike mRNA, miRNA is very short and can be easily synthesized; synthesized molecules with known concentration could thus be used as a reference for quantifying miRNA in a sample. As shown in Figure 1, sequence labels for discriminating the sources of miRNA (sample or reference) are designed into the loop near to the 3’-end of the miRNA-specific RT primer, so that the 5’ end of the primer can offer a universal priming site for the following PCR. The structure of the miRNA-specific stem–loop RT primer is the same as that used by Chen’s group. After reverse transcription with the sequence-tagged RT primers, cDNA from the different sources (sample and reference) were similarly labeled with different sequences (thus, different colors in a fluorescence-based assay). To avoid PCR-bias resulting from Tm differences, the labels were designed from the same base species but with different base order. We labeled the sample-miRNA and the reference-miRNA with the sequences “catg” and “gatc” respectively ; hence, in a pyrogram (Figure 1), [a] H. Jing, Prof. Q. Song, Z. Chen, B. Zou, Prof. G. Zhou Huadong Research Institute for Medicine and Biotechnics Nanjing 210002 (China) Fax: (+ 86) 25-8451-4223 E-mail : ghzhou@nju.edu.cn [b] Prof. Q. Song, B. Zou School of Life Science and Technology, China Pharmaceutical University Nanjing 210009 (China9 [c] C. Chen, Prof. M. Zhu Model Animal Research Centre, Nanjing University Nanjing 210093 (China) [d] Z. Chen, Prof. G. Zhou Medical School, Nanjing University Nanjing 210093 (China) [e] T. Kajiyama, Prof. H. Kambara Central Research Laboratory, Hitachi, Ltd. Tokyo 185-8601 (Japan) Supporting information for this article is available on the WWW under http ://dx.doi.org/10.1002/cbic.201100023.