Trizol rna extraction agent

Total RNA was extracted from endometrial cancer and nonneoplastic endometrial tissues using TRIzol RNA extraction agent (Invitrogen) according to the manufacturer's instructions 12 (link). Only RNA that resulted in an A260/280 ratio of 1.8-2.0 was reverse transcribed to generate cDNA. Synthesis of cDNA was carried out with SuperScript II RNase Reverse Transcriptase (Invitrogen) and primers (Invitrogen) at 42°C with 2µg of total RNA as template, in a final volume of 20µl. Negative controls for the reverse-transcription reaction were prepared by omitting the RT enzyme 13 (link), 14 (link). For the reverse transcription-PCR, the relative intensity of Na_v1.7 mRNA expression was measured by densitometry (ImageJ, Bethesda, USA). For conventional end-point PCR, 100 ng of cDNA was amplified following addition to a 30µl mastermix containing dNTPs, Platinum Taq DNA polymerase (Invitrogen) enzyme and appropriate forward and reverse primers for the desired Na_v α subunits target gene. Amplicons were visualized under UV light following separation through a 1% agarose gel containing ethidium bromide.

With the aid of the TRIzol RNA extraction agent (Invitrogen, 12,183,555, Shanghai, China), ribosome-nascent chain (RNC)-mRNA was isolated following with the instruction of test kits.