Hairpin ittm mirna qpcr quantitation kit

Following the experimental design, commercially available Lentiviral vectors were used to construct the LV2-hsa-miR-1254-mimic vector (miR-1254-mimic) and the LV2-hsa-miR-1254-inhibitor vector (miR-1254-inhibitor) (GenePharma, Shanghai, China). These structures were used to overexpress or knockdown miR-1254 in GC cells after being verified by DNA sequencing. The LV2 empty lentiviral construct (miR-NC) acted as a negative control. When MGC803 and SGC7901 grew to 40–50% confluence, they were infected by miR-1254-mimic, miR-1254-inhibitor, and miR-1254-NC at a suitable multiplicity of infection (MOI). Stable cell lines were obtained by using 5 μg/ml puromycin (Sigma, Aldrich) for about a week. Then we used Hairpin-itTM miRNA qPCR Quantitation Kit (GenePharma, China) to analyze the miR-1254 expression of cells. Lentiviral vector containing Smurf1 and shRNA coding sequence (LV-Smurf1, Smurf1-shRNA) were also constructed by Genepharma Biotech (Shanghai, China). The scrambled lentiviral construct was performed as a negative control. Stable cells were generated by means of the above procedure. Then we used qRT-PCR and western blot to analyze the expression of Smurf1.

Total RNA from mammary gland tissues of 15 cows was synthesized into cDNA using the NCode™ miRNA First-Strand cDNA Synthesis kit (Invitrogen, USA), and miRNA expression was measured using a Hairpin-it^TM miRNA qPCR Quantitation Kit (GenePharma, Suzhou, China) according to the manufacturer’s protocol. Alternatively, total RNA was synthesized into cDNA using a cDNA Synthesis kit (Exiqon, USA) to measure mRNA expression using SYBR® Green Master Mix (Exiqon, USA) according to the manufacturer’s protocol. The calculation of relative expression levels of selected miRNAs or mRNAs was conducted using the ΔΔCt method^{86 (link)}, with U6 or β-actin as an internal control. The experiment was performed in triplicate. Statistical comparisons were performed with unpaired two-tailed T-tests. Differences were considered significant for an adjusted P value ≤ 0.05.

The total RNA was extracted from mouse liver, lung, spleen, kidney, brain tissue (stored at −80°C) using TRIzol reagent (Invitrogen) following the manufacturer's instructions. RNA purity and concentration were determined using a Multiskan™ FC reader (Thermo Fisher Scientific, USA). For miR-100 analysis, the cDNA was synthesized with random primers using a reverse transcription kit (Gene Pharma, Shanghai, China). Levels of mature miR-100 were measured using a Hairpinit TM miRNA qPCR Quantitation Kit (Gene Pharma, Shanghai, China) according to manufacturer's protocol. The small nuclear RNA U6 served as an internal control. For mRNA analysis, the cDNA was synthesized using a PrimerScript™ RT reagent kit (TaKaRa, China) and random primers (Sangon Biotech, Shanghai, China). Real-time PCR was performed using TB Green™ Premix Ex Taq™ II (TaKaRa, China) according to the manufacturer's instructions. β-actin was used as an internal control. The relative expression of genes was analyzed utilizing the 2^−ΔΔ Ct method. The sequences of primers were listed in Table 2.