Miscript syber green pcr kit

Total RNA including miRNA was extracted from serum samples using miRNeasy Mini kit (Qiagen, Germany). Frozen serum samples were incubated at 37 °C in a water bath until they were completely thawed, then 200 μL of serum were added to 1000 μl of Qiazol lysis reagent. The extraction was performed according to the instruction manual of the manufacture. cDNA was synthesized from the isolated RNA using miScript II RT kit (Qiagen) according to the manufacturer instructions. The relative expression of miRNAs was quantified by miScript SYBER Green PCR kit (Qiagen), which includes miScript universal primer and quantiTect SYBER Green PCR master mix. The reactions were performed in 384 well plates with 10 μL total volume/well and contained 1X SYBER green master mix, 200 nmol/L forward primer (miRNA specific primer) and 200 nmol/L universal primer, using 3 ng cDNA/well. The conditions included initial denaturation at 95 °C for 15 min., followed by 40 cycles of 94 °C for 15 s, 55 °C for 30 s, and 72 °C for 30 s. All the samples were performed in duplicates on ViiA 7 real time PCR system (Applied Biosystem, Foster city, CA, USA). miR-16 was used as an endogenous control (25 (link), 26 (link)). The relative gene expression analysis of the target miRNAs was performed using the delta-delta-Ct method as described previously (27 ).

RNA was extracted from the samples using TRIZOL (Sigma-Aldrich) according to the manufacturer's protocol. cDNA was synthesized from the extracted RNA using high-capacity cDNA reverse transcription kit (Applied Biosystems, Austin, TX, USA) in accordance with the manufacturer's instructions. Real-time detection was performed with SYBER Green master mix (Applied Biosystems) using following primers for TRIM32 and GAPDH: TRIM32 forward 5′-GGCCACTGTACACTCCCTGT-3′ and TRIM32 reverse 5′-AGCCAAAGAGCCTGTGAAGA-3′, GAPDH forward 5′-GAAGGTGAAGGTCGGAGTC-3′ and GAPDH reverse 5′-GAAGATGGTGATGGGATTTC-3′. The cycling conditions used were 95 °C for 3 min (1 cycle), 95 °C for 20 s, 62 °C for 30 s and 72 °C for 40 s (35 cycles).
For miRNA isolation, RNA was isolated from the cells using miRNAeasy mini kit (Qiagen, Hilden, Germany). cDNA was synthesized using miScript II RT kit (Qiagen) according to the manufacturer's protocol. Specific primers for miR-155-5p and Let-7a-5p (Qiagen) were used to detect the respective miRNAs. qPCR was performed to detect the levels of miRNAs of interest using miScript SYBER Green PCR kit (Qiagen) as per the manufacture's protocol. qPCR was carried out using ROTOR-GENE Q (Qiagen). Specificity of all the primers used was confirmed with single product peak in the melt curve analysis.