MicroRNAs (miRNAs) are endogenous ~22 nt RNAs that play important regulatory roles in animals and plants by targeting mRNAs for cleavage or gene silencing. Although quantitative real-time PCR (qRT-PCR) had been widely used for miRNAs quantification, a multiplex quantification method is demanding. In this study, we successfully detected 2 miRNAs (miR-505-3p and miR-21a-5p) and an internal control (miR-16-5p) with only one reaction based on competitive PCR (cPCR) with high sensitivity. For each miRNA, two stem-loop reverse transcription (RT) primers were designed to produce two different templates: the competitor cDNA and the target cDNA, which had similar sequences except for 3 nucleotides different in length. RNA from a control sample was reverse transcribed with the competitive RT primers of multiple genes. Samples for test were reverse transcribed with target RT primers to obtain target cDNAs. Target cDNA was mixed with competitor cDNA to be used as the template for a multiplex fluorescent cPCR reaction. The cPCR products were separated on polyacrylamide gel electrophoresis with ABI 377 DNA sequencer and each fluorescent peak was quantified by its intensity. In this method, we compared the expression level of miR-505-3p in two tissues (thalamus and tail) between C57BL/6J and C3H/HeJ mice. The results showed that in the thalamus, which had high abundance of miR-505-3p, both cPCR and SYBR Green based qRT-PCR provided a sensitive quantification outcome. However, in the tail, which had extremely low level of miR-505-3p, it could be steadily detected by cPCR even after 8 times dilution with a relatively high sensitivity, while qRT-PCR can’t detect any product only after 2 times dilution. The variation as low as 12.2% between samples could be clarified by cPCR, which could not be accomplished by qRT-PCR. This method enables multiplex, accurate and sensitive quantification of miRNAs with fewer precious RNA samples than qRT-PCR.

Understanding of the functional significance of microRNAs (miRNAs) requires efficient and accurate detection method. In this study, we developed an improved miRNAs quantification system based on quantitative real-time polymerase chain reaction (qRT-PCR). This method showed higher efficiency and accuracy to survey the expression of primary miRNAs (pri-miRNAs), precursor miRNAs (pre-miRNAs), and mature miRNAs. Instead of relative quantification method, we quantified the pri-miRNAs and pre-miRNAs with absolute qRT-PCR based on SYBR Green I fluorescence. This improvement corrected for the inaccuracy caused by the differences in amplicon length and PCR efficiency. We also used SYBR Green method to quantify mature miRNAs based on the stem–loop qRT-PCR method. We extended the pairing part of the stem–loop reverse transcript (RT) primer from 6 to 11 bp, which greatly increased the efficiency of reverse transcription PCR (RT-PCR). The performance of the improved RT primer was tested using synthetic mature miRNAs and tissue RNA samples. Results showed that the improved RT primer demonstrated dynamic range of seven orders of magnitude and sensitivity of detection of hundreds of copies of miRNA molecules.

Cell-specific DNA methylation pattern detection is of great importance for the tumorigenesis and differentiation studies. Comparatively, large amounts of DNA were needed for traditional methods of DNA methylation pattern detection, and therefore, more sensitive method for high throughput analysis with a limited amount of DNA is needed. With Mouse 3T3 cells, we developed new multiplex-nested PCR technologies for bisulfite-assisted genomic sequencing PCR (BSP) methylation pattern detection method. Primers step add-in method and templates precipitation methods efficiently increase the throughput of the assay, and the nested PCR method also increased the sensitivity. The optimized assay could successfully detect 15 sequences of methylation pattern with a minimal amount of DNA (500–1,000 cells of genome DNA).

Microtus fortis is a special resource of rodent in China. It is a promising experimental animal model for the study on the mechanism of Schistosome japonicum resistance. The first complete mitochondrial genome sequence for Microtus fortis calamorum, a subspecies of M. fortis (Arvicolinae, Rodentia), was reported in this study. The mitochondrial genome sequence of M. f. calamorum (Genbank: JF261175) showed a typical vertebrate pattern with 13 protein coding genes, 2 ribosomal RNAs, 22 transfer RNAs and one major noncoding region (CR region).The extended termination associated sequences (ETAS-1 and ETAS-2) and conserved sequence block 1 (CSB-1) were found in the CR region. The putative origin of replication for the light strand (OL) of M. f. calamorum was 35 bp long and showed high conservation in stem and adjacent sequences, but the difference existed in the loop region among three species of genus Microtus. In order to investigate the phylogenetic position of M. f. calamorum, the phylogenetic trees (Maximum likelihood and Bayesian methods) were constructed based on 12 protein-coding genes (except for ND6 gene) on H strand from 16 rodent species. M. f. calamorum was classified into genus Microtus, Arvcicolinae for the highly phylogenetic relationship with Microtus kikuchii (Taiwan vole). Further phylogenetic analysis results based on the cytochrome b gene ranged from M. f. calamorum to one of the subspecies of M. fortis, which formed a sister group of Microtus middendorfii in the genus Microtus.The organization of the M. f. calamorum mitochondrial genome. The tRNAs were denoted using single letter amino acid code. The protein coding genes were indicated as follows: ND1-6, NADH dehydrogenase subunits 1–6; COX1–3, cytochrome c oxidase subunits 1–3; ATP6 and ATP8, ATPase subunits 6 and 8; Cyt b, Cytochrome b; and CR, control region.Download high-res image (118KB)Download full-size imageHighlights► The first mitochondrial genome of M. f. calamorum was reported in this study. ► The mitochondrial genome of M. f. calamorum showed a typical vertebrate pattern. ► M. f. calamorum show highly phylogenetic relationship with Microtus kikuchii. ► M. f. calamorum formed a sister group with Microtus middendorfii in the genus Microtus.