•A breathing-based isothermal emulsion amplification (BIEA) method was developed.•BIEA showed excellent properties compared with conventional amplification method.•Terminal breathing of DNA duplex was firstly used in emulsion amplification.The reaction temperature is one of the main factors that affect the stability of emulsion PCR (emPCR). Focusing on this point, we applied the “DNA breathing” mechanism in BEAMing (Bead, Emulsion, Amplification, and Magnetic) and proposed a more stable emulsion amplification method. Compared to the conventional emPCR, this method provided excellent results. Firstly, more stable emulsion system resulted in higher percentage of single-molecular amplifications (73.17%). Secondly, an ordinary temperature-controlling device was enough. Our outcome showed that the reaction temperature of this method was not strict so that the ordinary temperature-controlling device was enough for it (the heat block sets vs. the PCR instrument: 13.140 ± 0.110 vs. 13.008 ± 0.039, P = 0.120). Thirdly, the single-biotinylated emP1 coated streptavidin beads were stable enough to be used for this method (the control temperature vs. the reaction temperature: 2967.91 ± 409.045 vs. 3026.22 ± 442.129, P = 0.334), which could replace the double-biotinylated emP1 coated beads and was benefit for saving cost. In conclusion, the method presented here with stable emulsion system, simplified temperature-controlling device, and decreased investment would be a highly streamlined and inexpensive option for future single-molecular amplification based researches.Download high-res image (202KB)Download full-size image

•The amplification-based technology with significant advantage in higher sensitivity is a key component of miRNA studies.•Advanced amplification-based methods cannot replace traditional ones to be conventionally used due to the lack of enough verification.•Several sample-specific normalizers have been validated, suggesting that a single universal normalizer for all sample types is unlikely to exist, yet the use of different combinations of several normalizers for different sample types may be more appropriate.Over the last two decades, the study of miRNAs has attracted tremendous attention since they regulate gene expression post-transcriptionally and have been demonstrated to be dysregulated in many diseases. Detection methods with higher sensitivity, specificity and selectivity between precursors and mature microRNAs are urgently needed and widely studied. This review gave an overview of the amplification-based technologies including traditional methods, current modified methods and the cross-platforms of them combined with other techniques. Many progresses were found in the modified amplification-based microRNA detection methods, while traditional platforms could not be replaced until now. Several sample-specific normalizers had been validated, suggesting that the different normalizers should be established for different sample types and the combination of several normalizers might be more appropriate than a single universal normalizer. This systematic overview would be useful to provide comprehensive information for subsequent related studies and could reduce the un-necessary repetition in the future.

Circulating miRNAs have been identified as key regulators of gene expression in many physiological and pathological processes. Real-time quantitative PCR is a conventional method that is indispensable, although many different approaches have been employed for miRNA expression profiling. We have established a universal, sensitive, highly efficient, cost effective and time-saving reverse transcription quantitative PCR for the measurement of circulating miRNA. This method involves the use of a random pre-adenylated DNA oligonucleotide linked to miRNAs followed by a universal reverse transcription and individual miRNA quantitative process. This method was optimized, and its specificity and sensitivity were evaluated. Circulating miRNAs from lung cancer patients were detected for verification. The results suggest that this random pre-adenylated DNA oligo-based miRNA quantitative method is sufficiently efficient and sensitive to detect circulating miRNA.

Deletions in Y chromosome are thought to be pathologically involved in some cases of male infertility associated with azoospermia or oligozoospermia. An emulsion-based multiplex PCR method was developed for detecting Y chromosome microdeletions in infertile men and a plasma sample of pregnant women carrying a male fetus. The sensitivity of multiplex PCR in emulsion was evaluated. Conventional PCR was also carried out for comparison. A total of 13 sequence-tagged sites (STSs) distributed in the AZF region were analyzed simultaneously with this method. The SRY gene was also detected as the inner control. Results showed that Y chromosome microdeletions were found in 4 of 19 infertile patients. Also, in 1 of 63 samples collected from pregnant women, microdeletions were found in some of the detected sites. It is suggested that the emulsion PCR assay was proven to be a promising diagnostic tool and could be widely used in further clinical and academic research.

ObjectiveTo profile the differential expression of plasma miRNAs in gestational diabetes mellitus (GDM).MethodsIn a pilot study conducted at a tertiary hospital in China between 2010 and 2014, peripheral blood samples were collected from women at 16–19 weeks of pregnancy. Pooled samples from 10 women who were subsequently diagnosed with GDM and from 10 healthy controls were used to construct two small RNA libraries. High-throughput sequencing was performed, and differentially expressed miRNAs were validated by quantitative real-time polymerase chain reaction (qRT-PCR), followed by target prediction, Gene Ontology analysis, and pathway identification.ResultsSequencing revealed 32 miRNAs that were differentially expressed in GDM, including 12 miRNAs that were upregulated and 20 that were downregulated. Differential expression of five upregulated miRNAs (hsa-miR-16-5p, hsa-miR-17-5p, hsa-miR-19a-3p, hsa-miR-19b-3p, hsa-miR-20a-5p) was confirmed by qRT-PCR. Target prediction showed that the major targets of these miRNAs were associated with insulin resistance and abnormal pregnancies.ConclusionThe five miRNAs that were differentially expressed in GDM could serve as noninvasive biomarkers. The results also provide insights into the molecular mechanisms that underlie GDM, thereby contributing to the diagnosis and treatment of this disease.

•We found that miR-181b-5p was up-regulated in SCC; miR-486-5p was down-regulated in AC from serum and tissue samples of NSCLC based on NGS.•The targets were negatively regulated by these two miRNAs based on qRT-PCR.•The pathway of miRNAs and targets were predicted.•Our study suggested that miR-181b-5p and miR-486-5p could be potential biomarkers for early diagnosis of NSCLC.BackgroundLung cancer is the leading cause of cancer deaths in China. Non-small cell lung cancer (NSCLC) is the major type of lung cancer.ObjectivesThe aim of our study was to characterize the expression profiles of miRNAs in serum and tissue of NSCLC at the same time, and to find more accurate relationship of miRNAs between serum and tissue. Furthermore, we intended to find more biomarkers of miRNAs in NSCLC samples.MethodsIn this study, the miRNAs were sequenced in 18 paired serum and 18 paired tissue samples. The expression levels of miRNAs and targets were quantified by qRT-PCR. The function analysis was performed by using bioinformatics methods.ResultsIn these paired samples miR-181b-5p was up-regulated in squamous cell carcinoma (SCC), miR-486-5p was down-regulated in adenocarcinoma (AC), and miR-21-5p was up-regulated in both SCC and AC. However, miR-181b-5p and miR-486-5p were rarely reported in lung cancer related studies. The expression levels of these two miRNAs and their targets, RASSF1 and PIK3R1 were quantified in additional samples by qRT-PCR. The results showed that the targets were negatively regulated by the two miRNAs. In addition, we noted that RASSF1 and PIK3R1 were directly involved in non-small cell lung cancer pathway.ConclusionsOur study suggested that miR-181b-5p and miR-486-5p could be new potential biomarkers for early diagnosis of NSCLC.

Circulating miRNAs have been identified as key regulators of gene expression in many physiological and pathological processes. Real-time quantitative PCR is a conventional method that is indispensable, although many different approaches have been employed for miRNA expression profiling. We have established a universal, sensitive, highly efficient, cost effective and time-saving reverse transcription quantitative PCR for the measurement of circulating miRNA. This method involves the use of a random pre-adenylated DNA oligonucleotide linked to miRNAs followed by a universal reverse transcription and individual miRNA quantitative process. This method was optimized, and its specificity and sensitivity were evaluated. Circulating miRNAs from lung cancer patients were detected for verification. The results suggest that this random pre-adenylated DNA oligo-based miRNA quantitative method is sufficiently efficient and sensitive to detect circulating miRNA.