Tumor tissues were collected simultaneously. All the resected samples were placed immediately into liquid nitrogen and stored at - 80°C.
Extracted molecule
total RNA
Extraction protocol
RNA was extracted by RNA isolation kit (Cat.No.83913, Sigma-Aldrich) per the manufacturer's protocol. 250 ng amount of RNA was reverse transcribed to cDNA by a Reverse Transcription Reaction kit (Qiagen, Hilden, Germany).
Label
SYBR Green
Label protocol
RT2 miRNA qPCR Assay were performed using the Custom RT2 PCR Arrays (QIAGEN, Hilden, Germany) following the Manufacturer’s instructions. 400ng RNA was reverse transcribed to cDNA in 20μl system by First Strand cDNA Synthesis Kit for RT-PCR (Merck & Co., Inc., Kenilworth, NJ, USA). Real-time PCR was performed using the Mx3000P real-time PCR system (Thermo Fisher Scientific, Waltham, MA, USA). PCR was carried out as follows: 40 cycles of 94 °C for 15s, 60 °C for 10s and 72 °C for 20s.
Hybridization protocol
n/a
Scan protocol
n/a
Description
Control
Data processing
The normalization and all the data analysis were performed according to the manufacturers instructions using their web-based software package: https://geneglobe.qiagen.com/cn/analyze/ For the normalization it uses the average of five housekeeping genes: SNORD61, SNORD68, SNORD72, SNORD95, SNORD96A. Target gene signals normalized to housekeeping genes; 2^-deltaCt, where deltaCt = (Ct_Target − Ct_HKG)] The web-based software package automatically performs all deltadeltaCt based fold-change calculations from the uploaded raw threshold cycle data.
