Improve Research Reproducibility A Bio-protocol resource
Concise Method
tooltip

Real-time PCR (qRT-PCR) and RNase R treatment

ZL Zhiping Lin
PL Peng Li
YT Yangyang Tang
HT Hongchang Tan
LL Lianxiang Luo
ask Ask a question
Favorite

TRIzol reagent (Invitrogen) was utilized to extract total RNA from 38 cartilage tissues (21 OA and 17 healthy control samples) and 2 chondrocyte cell lines. To detect the expression of hsa_circ_0007292 and HMGB1, cDNA was synthesized using M-MLV Reverse Transcriptase (Invitrogen) from 1 μg of total RNA. After preparing PCR reaction systems with SYBR® Green (Promega, Madison, WI, USA), qRT-PCR was performed on ABI 7500 System with GAPDH as an endogenous control. For miRNAs analysis, TaqMan® MicroRNA Reverse Transcription Kit and TaqMan® Universal Master Mix II (Thermo Fisher, Waltham, MA, USA) were used and U6 was used as the endogenous reference. The relative gene expression was calculated by the 2−ΔΔCt method. The primer sequences were listed in Table ​Table11.

Primer sequences

For RNase R treatment, 2 μg of total RNA from cells were treated with 3 U/μg RNase R (Epicentre Technologies, Madison, WI, USA) for 30 min at 37 °C. The expression of hsa_circ_0007292 and its corresponding linear mRNA ATP5C1 expression was determined by qRT-PCR.

Copyright and License information: The Author(s) ©2023
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 licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence 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 licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.

Do you have any questions about this protocol?

Post your question to gather feedback from the community. We will also invite the authors of this article to respond.

post Post a Question
0 Q&A