Improve Research Reproducibility A Bio-protocol resource
Concise Method
tooltip

Reverse transcription PCR and quantitative PCR analysis

EH Etienne Humblin
MT Marion Thibaudin
FC Fanny Chalmin
VD Valentin Derangère
EL Emeric Limagne
CR Corentin Richard
RF Richard A. Flavell
SC Sandy Chevrier
SL Sylvain Ladoire
HB Hélène Berger
RB Romain Boidot
LA Lionel Apetoh
FV Frédérique Végran
FG François Ghiringhelli
request Request a Protocol
ask Ask a question
Favorite

Total RNA from T cells was extracted using Trizol (Invitrogen, Carlsbad, CA, USA). In total, 300 ng of RNA were reverse-transcribed into cDNA using M-MLV reverse transcriptase (28025-013, Invitrogen), Random Primers and RNAseOUT inhibitor (10777-019, Invitrogen). cDNA were quantified by real time PCR using a Power SYBR Green Real-time PCR kit (4367659, Life technologies) on a 7500Fast detection system (Life technologies). Relative mRNA levels were determined using the ∆Ct method. Values were expressed relative to β-actin unless otherwise specified. The sequences of the oligonucleotides used are described in Supplementary Table 1.

Copyright and License information: The Author(s) ©2017
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/.

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