Rever traace qpcr rt kit master mix with gdna remover

Total RNA was extracted and isolated from the cell lines and frozen tumor specimens using a AxyPrep Multisource Total RNA Miniprep Kit from Axygen (Corning, Suzhou, Jiangsu, China), and the first strand cDNA was synthesized using the Rever TraAce qPCR RT Kit Master Mix with gDNA Remover (FSQ-301, Toyobo Co. Ltd. Osaka, Osaka Prefecture, Japan) according to the manufacturer’s instructions. Quantitative real-time polymerase chain reaction (qRT-PCR) was performed as previously described [10 (link)]. Briefly, qRT-PCR (SYBR Green Assay, Roche Diagnostics GmbH, Indianapolis, IN, USA) was performed on a 7500 FAST Real-Time PCR System (Applied Biosystems). The relative expression levels of the mRNA were calculated and quantified using the 2^−ΔΔT method after normalization to the expression of the control. GAPDH served as the endogenous control. The primer sequences are described in Additional file 2: Table S2 and were purchased from Comate Bioscience (Institute of Biotechnology, Jilin, China).

The total RNA was extracted from cells and tissues with an AxyPrep Multisource Total RNA Miniprep Kit from Axygen (Corning, NY). The quality and concentration of RNA were determined with NANODROP 2000c Spectrophotometer (Thermo Fisher Scientific). To detect the expression of lncRNA and mRNA, reverse transcription was performed with the help of the Rever TraAce qPCR RT Kit Master Mix with gDNA Remover (FSQ-301, Toyobo Co., Ltd., Osaka, Osaka Prefecture, Japan). To detect the expression of miRNA, reverse transcription was performed by Mir-X™ miRNA First-Strand Synthesis kit (Clontech Laboratories, Mountain View, CA, USA). qRT-PCR was performed on a 7500 FAST Real-Time PCR System (Applied Biosystems) using SYBR Green Assay (Roche Diagnostics GmbH, Indianapolis, IN, USA). LncRNA and mRNA were normalized to GAPDH while miRNA to U6, respectively. The primer sequences are shown in Supplementary Table 3. The fold changes were calculated using relative quantification (the 2^−ΔΔCt method).