Rna isolation kit

RNA was harvested from indicated cells using TRIzol reagent (Invitrogen) and an RNA isolation kit (CWBIO, China) following the manufacturer’s instructions. Equal amounts of RNA were subjected to reverse transcription with M-MLV reverse transcriptase (Promega). The qRT-PCR experiment was performed using the SYBR master mixture (Takara) on a real-time PCR machine TP800 (Takara). The primer sequences are listed in Table 1. The expression of the targeted genes was normalized to GAPDH.

Total RNA was extracted from tumors using an RNA Isolation kit (CWbiotech Co., Ltd., Beijing, China) following the manufacturer's protocol. Stem-loop RT-qPCR for mature miR-21 was performed as previously described (28 (link)). RT-qPCR for PDCD4 was performed using Power SYBR^® Green PCR Master mix (Agilent Technologies, Inc., Santa Clara, CA, USA) in a final volume of 20 µl, comprising of 100 ng cDNA, 10 µl master mix, 1 µl ROX and 0.4 pmol/µl of each primer. qPCR cycling conditions were as follows: 95°C for 2 min, and then 95°C for 15 sec and 55°C for 30 sec, for 40 cycles, followed by 60°C for 1 min. The melting curve was 65–95°C. Human U6 mRNA was used for normalization for the stem-loop RT-qPCR and GAPDH was used for normalization for the PDCD4 RT-qPCR. Fluorescent signals were normalized to these internal reference genes, and the threshold cycle (Cq) was set within the exponential phase of the PCR. The relative gene expression was calculated by comparing cycle times for each target PCR. The target PCR Cq values were normalized by subtracting the U6 or GADPH Cq value, which provided the ΔCq value. The relative expression level between treatments was then calculated using the following equation: Relative gene expression=2^{(ΔCqsample−ΔCqcontrol)} (30 (link)).