Improve Research Reproducibility A Bio-protocol resource
Concise Method
tooltip

RNA extraction and RT-qPCR

NS Nickie Safarian
PW Paige Whyte-Fagundes
CZ Christiane Zoidl
JG Jörg Grigull
GZ Georg Zoidl
request Request a Protocol
ask Ask a question
Favorite

Total RNAs were extracted from 6 dpf larvae using RNeasy Plus Mini Kit (Qiagen). The iScript Reverse Transcription Supermix (Bio-Rad Laboratories, Mississauga, Canada) was used to reverse transcribe 1 µg of total RNA. The cDNA equivalent of 15 ng total RNA was analyzed in triplicate by quantitative Real Time-PCR using the SsoAdvanced SybrGreen PCR mix (Bio-Rad). All experiments included a melt curve analysis of PCR amplicons generated in each reaction. Raw cycle threshold values (Ct-values) were exported from the CFX Manager Software (Bio-Rad, Canada), and the relative gene expression was calculated using the Relative Expression Software Tool (REST-2009)66. Gene information and primer sequences are listed in Supplementary Table S4.

Copyright and License information: The Author(s) ©2020
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