Quantitect reverse transcription cdna synthesis kit

Total RNA was isolated from the cells with TRI reagent™ (Sigma) and reverse transcribed using QuantiTect Reverse Transcription cDNA synthesis kit (Qiagen, Hilden, Germany), according to the manufacturers' instructions. Quantitative RT-PCR analysis was carried out on the LightCyclerTM 1.0 (Roche) using QuantiTect SYBR Green PCR kit (Qiagen), according to the manufacturers' instructions as previously described [33] (link). Primers were specific to HER2, MUC4 or β-actin as normalizing gene (Table 1; Eurofins). Expression levels for each gene were quantified against serial dilutions of purified PCR product.

RNA extraction was performed using Qiagen RNA isolation kits following manufacturer’s instruction. 1 µg of RNA was reverse transcribed using the quantitect reverse transcription cDNA synthesis kit following manufacturer’s instruction (Qiagen, MD, USA).

RNAs were extracted from OV2008 and C13 cells treated with QC at 0, 5 and 10 µM using Qiagen RNA isolation kits following manufacturer’s instruction. About 1 μg of RNA was reverse transcribed using the QuantiTect Reverse Transcription cDNA synthesis kit (Qiagen, Germantown, MD, USA). Quantitative real-time PCR (qRT-PCR) was carried out using SYBR-Green PCR Master Mix (Applied Biosystems, Foster City, CA, USA) in CFX96 Real-Time PCR System (Bio-Rad, Hercules, CA, USA) as reported previously [18 (link)] by using primers synthesized from Integrated DNA Technologies (IDT). The sequences for the genes analyzed are CTSL FP:5′-CAATCAGGAATACAGGGAAGGG-3′, CTSL RP:5′-CTGGGCTTACGGTTTTGAAAG-3′, and GAPDH FP:5′-ACATCGCTCAGACACCATG-3′ and GAPDH RP:5′-TGTAGTTGAGGTCAATGAAGGG-3′.