PCR

Published: 23-07-2020| Version 1 | DOI: 10.17632/f87srd3bz6.1
Contributor:
wenjingzhang zhang

Description

We analyzed mRNA levels of FGF2 and SOX2 in and around esophageal squamous cell carcinoma. Furthermore, they are highly expressed in tumor tissues and promote tumor progression

Files

Steps to reproduce

Firstly, 29 cases of esophageal cancer and their paired adjacent normal tissues were taken out from the refrigerator at -80°C. Secondly, the liquid nitrogen was added to them for milling, and Trizol reagent was also added to extract the total RNA in the tissues after grinding according to the instructions. Thirdly, the concentration of total RNA in the extracted tissues was measured by NanoDrop 2000c uv spectrophotometer. After the electrophoresis test, cDNA was transcribed. According to the instructions of the reverse transcription kit,2ng taken from the total RNA extracted was added into the reverse transcription reaction system under the following conditions :25℃5min 42℃ 60min, 70℃ 5min and 4℃ forever. The synthesized cDNA was stored in a refrigerator at -80℃ for later use. The primer sequences are listed in Table 2. qRT-PCR use a two-step method with SYBR Green (Applied Biosystems 7500 ,ThermoFisher Scientific);The above reaction elements were added into the reaction system, and the reaction conditions were as follows: pre-denaturation at 95°C for 2 min, denaturation at 95°C for 10 S, annealing at 60°C for 10 S, extension at 72°C for 5 min, 40 cycles in total. Each sample was repeated at least 3 times, with 3 multiple holes set for each time.