Mysticq microrna qpcr assay primer

TRIzol reagent (Invitrogen) and mirVana miRNA Isolation Kit (Thermo, Shanghai, China) were employed to extract the total RNAs and total miRNAs, respectively. The first cDNA chains of mRNA and miRNA were synthesized by the Prime Script RT Reagent Kit (Takara Biotechnology Co., Ltd., Dalian, China) and the miRNA Reverse Transcription Kit (Life Technologies, Foster, CA, USA), respectively. The qPCR was performed by using the SYBR Premix Ex Taq (Takara) and MystiCq microRNA qPCR Assay Primer (Sigma, Missouri, USA) on Applied Biosystems StepOnePlus™ Real-Time PCR System. The normalization for miR-193a and PIK3CG was used as U6 and GAPDH, respectively. The primers were miR-193a F: 5′-ACTGGCCTACAAAGTCCCAGT-3′, R: 5′-GTGCAGGGTCCGAGGT-3'; U6 F: 5′-CTTCGGCAGCACATATAC-3′, R: 5′-GAACGCTTCACGAATTTGC-3'; PSEN1 F: 5′-TATGGCAGAAGGAGACCCG-3′, R: 5′-CCATTCCTCACTGAACCCG-3'; GAPDH F: 5′-AAGGTGAAGGTCGGAGTCAA-3′, R: 5′-AATGAAGGGGTCATTGATGG-3'.

miRNA expression profiles in the HCC and matched control tissues were examined in a miRNA array (Shanghai Wcgene Biotech, Shanghai, China) using high-throughput quantitative RT-PCR (qRT-PCR). Total RNA was isolated from tissue samples, purified, polyadenylated in a poly (A) polymerase reaction, and then reversed-transcribed into cDNA. Individual miRNAs were quantified with specific MystiCq microRNA qPCR assay primers (Sigma, St. Louis, USA).

Based on the miRNome miRNA array, we identified three signature miRNAs (miR-21-5p, miR-144-3p, and miR-let-7b-5p) that could have potential for early diagnosis of leptospirosis. We validated the efficacy of these miRNAs using patients’ serum samples. A serum-plasma RNA purification minikit (Norgen Biotek Corp., Canada) was used for the extraction of RNA. The RNA was reverse transcribed using a miScript II RT kit (Qiagen, Hilden, Germany), according to the manufacturer’s protocol. cDNA obtained from the RT reaction was diluted 1:10 with RNase-free water, and real-time qPCR was performed using the miScript SYBR green PCR kit (Qiagen, Hilden, Germany) that contains the miScript universal primer (reverse primer). MystiCq microRNA qPCR assay primers specific for miR-21-5p, miR-144-3p, and miR-let-7b-5p (Sigma-Aldrich, St. Louis, MO, USA) were used as forward primers. miRNA expression was normalized to the internal control SNORD25 (Sigma-Aldrich, St. Louis, MO, USA). Real-time qPCRs were performed at 95°C for 15 min, followed by 40 cycles of 94°C for 15 s, 60°C for 30 s, and 70°C for 30 s. Each reaction was carried out in triplicate in CFX96 detection system (Bio-Rad, Hercules, CA, USA), and data normalization was performed using the ΔΔC_T method.

The genes studied for their relative genetic expression patterns are provided in S1 Table. MystiCq microRNA SYBR Green qPCR MasterMix (Sigma Aldrich) was used to analyze 100 ng of cDNA from each experimental condition with MystiCq microRNA qPCR Assay primers and MystiCq Universal PCR primer (Sigma Aldrich). Quantitative real time PCR was performed using the StepOnePlus Real-Time PCR System. Data were analyzed using comparative CT method with internal control SNORD44 levels to normalize differences in sample loading. Results (mean ± standard deviation of triplicate reaction wells) represent the n-fold difference of microRNA transcript levels in a particular sample compared with samples of control Ishikawa cells.