Two step primescript mirna cdna synthesis kit

Total RNA was extracted from untreated cells and from cells treated with PLA with TRIzol reagent (Invitrogen) and was then incubated with DNase I. A 0.5 µg sample of total RNA was used for cDNA synthesis with a Two-Step PrimeScript miRNA cDNA Synthesis Kit (Takara, Dalian, China) and an ABI 7500 Real-Time PCR system (Applied Biosystems, Foster City, CA, USA). qRT-PCR was carried out with a SuperReal PreMix Plus (SYBR Green) Kit (TIANGEN Biotech Co., Ltd, Beijing, China) using a Light Cycler system. The relative RNA expression level after normalization to GAPDH was calculated according to the change in expression using the equation 2^−ΔΔCt. The primers used for the qRT-PCR analysis are listed in Additional file 1: Table S1.

Total RNA including microRNAs was extracted from culture cells using Trizol reagents and treated with DNaseI. qRT-PCR were performed using the Two-Step PrimeScript miRNA cDNA Synthesis Kit (Takara, Dalian, China) using a ABI 7500 Real Time PCR system (Applied Biosystems, Foster City, CA, USA). Relative miRNA expression levels after normalization to U6 small nuclear RNA were calculated using 2 [(Ct of miR-939) (Ct of U6)]. qRT-PCR was performed by SYBR Kit (Qiagen, China) using a Light Cycler system. Relative mRNA expression levels were normalized to the geometric mean of β-actin to control for variability in expression levels and calculated.