RNA extraction and quantitative qRT-PCR assays

Huaiming Sang; Weifeng Zhang; Lei Peng; Shuchun Wei; Xudong Zhu; Keting Huang; Jiajia Yang; Meihong Chen; Yini Dang; Guoxin Zhang

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RNA extraction and quantitative qRT-PCR assays

HS Huaiming Sang

WZ Weifeng Zhang

LP Lei Peng

SW Shuchun Wei

XZ Xudong Zhu

KH Keting Huang

JY Jiajia Yang

MC Meihong Chen

YD Yini Dang

GZ Guoxin Zhang

This method is extracted from research article: Cell Death Dis, Jan 2022

Exosomal circRELL1 serves as a miR-637 sponge to modulate gastric cancer progression via regulating autophagy activation

DOI: 10.1038/s41419-021-04364-6

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Total RNA from tissues and cells lines was extracted using TRIzol reagent (Invitrogen, USA) and NanoDrop 2000 spectrophotometer (Thermo Scientific, USA) was conducted to accomplish RNA quantity control and concentration detection. RNase R (Epicentre Biotechnologies, USA) experiment (15U) was applied on total RNA (5 ug) for 15 min at 37 °C. Transcription Kit (TaKaRa, RR036A, Japan) was then utilized to reverse the RNAs into cDNA and subsequently applied qRT-PCR on the LightCycler480II (Switzerland) to estimate the expression of mRNA or miRNA. MRNA results were normalized to the expression of GAPDH, while miRNA normalized to U6, and both were calculated to fold changes using 2(−ΔΔCT). The primers involved in this study were listed in Supplementary Table 3.

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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