S. A miRNA expression map was created with the new assay by detecting the expression of 3 301353-96-8 manufacturer miRNAs in four BARBL/c mouse tissue samples (n = 5). After measuring the expression of the small nuclear RNA (snRNA) U6 as a housekeeping gene, the miRNA data were normalized by calculating the relative 22DDCq value. The expression patterns of the 3 miRNAs were in agreement with the previous observations. miR-122 and miR133a were highly tissue specific and were expressed in the liver and the heart, respectively. miR-122 accounted for the domain of all mouse miRNAs found in the liver; miR-122 was almost undetected in all other tissues analyzed. miR-133a was expressed predominantly in the heart, and expressed at a low level in the liver. Additionally, let-7a acted as a housekeeping microRNA, and it was detected in all four mouse tissues (Figure 6).PD-1/PD-L1 inhibitor 1 DiscussionThe miRNAs play a crucial role in several biological processes and act as regulators of development, differentiation [19] and cell survival [6?]. miRNAs function by pairing with mRNAs ofprotein-coding genes and regulating their post-transcriptional expression [3]. A great number of studies have indicated that miRNA expression profiles classify human cancers [20,21]. Microarray is the most widely used high-throughput technique for the identification of a cancer-specific miRNA expression profile, but the low level of sensitivity is a disadvantage of this technique as it is difficult to amplify miRNA targets and can lead to false positive signals from closely related miRNAs and genomic sequences [22]. RT-qPCR is a powerful technique for quantifying gene expression in the life sciences and medicine as it is highly sensitive, accurate and simple [23]. And it is the most adaptive technique for the quantification of miRNAs 18055761 used by the general research community. Several RT-qPCR assays have been established for miRNA quantification, and are currently made available by companies. Considering the similar small size of miRNAs with ordinary PCR primers, we proposed an optimal and convenient alternative process based on readily available techniques and materials. This assay allowed reverse transcription of the entire miRNA population from the same sample with the identical level of efficiency, followed by the amplification and quantification of cDNA with the simple, high-throughput SYBRH Green real-time PCR without using fluorochromic hybridization probes or LNAmodified oligonucleotides. The expression of a considerable number of human 
miRNAs have been detected using thisFigure 4. Specificity and cross-reaction analysis of the miRNA assay. (A B) Relative level of PCR products using a mismatched primer compared to the perfectly matched primer in the normal program (annealing temperature 55uC) and in the high-stringency program (annealing temperature 62uC) for amplifying 
the target hsa-miR-32. Each column represents the mean (6 SD) of three measurements. (C D) Cross-reaction of the human let-7 family assays (annealing temperature 56uC). The percentage of cross-reaction values was calculated based on the Cq difference between assay-specific and nonspecific miRNA targets. The PCR annealing temperature was raised to 60uC to distinguish let-7a and let-7c. doi:10.1371/journal.pone.0046890.gFacile and Specific Assay for Quantifying MicroRNAFigure 5. Advantage of U-tailing of miRNA. Both mRNA and miRNAs showed no significant difference of Cq value and dissociation curve based on multiplex or single RT reactions (P.0.05.S. A miRNA expression map was created with the new assay by detecting the expression of 3 miRNAs in four BARBL/c mouse tissue samples (n = 5). After measuring the expression of the small nuclear RNA (snRNA) U6 as a housekeeping gene, the miRNA data were normalized by calculating the relative 22DDCq value. The expression patterns of the 3 miRNAs were in agreement with the previous observations. miR-122 and miR133a were highly tissue specific and were expressed in the liver and the heart, respectively. miR-122 accounted for the domain of all mouse miRNAs found in the liver; miR-122 was almost undetected in all other tissues analyzed. miR-133a was expressed predominantly in the heart, and expressed at a low level in the liver. Additionally, let-7a acted as a housekeeping microRNA, and it was detected in all four mouse tissues (Figure 6).DiscussionThe miRNAs play a crucial role in several biological processes and act as regulators of development, differentiation [19] and cell survival [6?]. miRNAs function by pairing with mRNAs ofprotein-coding genes and regulating their post-transcriptional expression [3]. A great number of studies have indicated that miRNA expression profiles classify human cancers [20,21]. Microarray is the most widely used high-throughput technique for the identification of a cancer-specific miRNA expression profile, but the low level of sensitivity is a disadvantage of this technique as it is difficult to amplify miRNA targets and can lead to false positive signals from closely related miRNAs and genomic sequences [22]. RT-qPCR is a powerful technique for quantifying gene expression in the life sciences and medicine as it is highly sensitive, accurate and simple [23]. And it is the most adaptive technique for the quantification of miRNAs 18055761 used by the general research community. Several RT-qPCR assays have been established for miRNA quantification, and are currently made available by companies. Considering the similar small size of miRNAs with ordinary PCR primers, we proposed an optimal and convenient alternative process based on readily available techniques and materials. This assay allowed reverse transcription of the entire miRNA population from the same sample with the identical level of efficiency, followed by the amplification and quantification of cDNA with the simple, high-throughput SYBRH Green real-time PCR without using fluorochromic hybridization probes or LNAmodified oligonucleotides. The expression of a considerable number of human miRNAs have been detected using thisFigure 4. Specificity and cross-reaction analysis of the miRNA assay. (A B) Relative level of PCR products using a mismatched primer compared to the perfectly matched primer in the normal program (annealing temperature 55uC) and in the high-stringency program (annealing temperature 62uC) for amplifying the target hsa-miR-32. Each column represents the mean (6 SD) of three measurements. (C D) Cross-reaction of the human let-7 family assays (annealing temperature 56uC). The percentage of cross-reaction values was calculated based on the Cq difference between assay-specific and nonspecific miRNA targets. The PCR annealing temperature was raised to 60uC to distinguish let-7a and let-7c. doi:10.1371/journal.pone.0046890.gFacile and Specific Assay for Quantifying MicroRNAFigure 5. Advantage of U-tailing of miRNA. Both mRNA and miRNAs showed no significant difference of Cq value and dissociation curve based on multiplex or single RT reactions (P.0.05.
