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RNA extraction, cDNA synthesis, and qRT-PCR

RK Rayyan Khan
XM Xinghua Ma
SS Shahen Shah
XW Xiaoying Wu
AS Aaqib Shaheen
LX Lixia Xiao
YW Yuanhua Wu
SW Shusheng Wang
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The total RNA was extracted from three biological repeats of drought-hardened and non-drought-hardened tobacco leaves of H and Y varieties using MiniBEST Plant RNA Extraction Kit (TaKaRa, Japan), according to the manufacturer instructions. The concentration of RNA was determined by Nanophotometer P330 (Implen, Munich, Germany) and its quality was assessed by agarose gel electrophoresis. After the extraction of RNA, the cDNA was synthesized via the PrimeScript 1st Strand cDNA Synthesis kit (TaKaRa, Japan), following the manufacturer protocol. The qRT-PCR was subsequently performed on Applied Biosystems QuantStudio3 real-time PCR machine (Applied Biosystems) on a total reaction volume of 20 μL using SYBER Green Master Mix (TaKaRa, Shiga, Japan). The qRT-PCR data were analyzed using the 2-△△CT method [98]. The gene-specific primers (Online Resource 2) were designed using PRIMER3 (https://www.ncbi.nlm.nih.gov/tools/primer-blast/). The Actin was used as a reference internal control gene. The raw data for qRT-PCR analysis was presented in Data Set 3 (supplementary data).

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

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