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Quantitative RT-PCR

CN Carme Nuño-Cabanes
VG Varinia García-Molinero
MM Manuel Martín-Expósito
MG María-Eugenia Gas
PO Paula Oliete-Calvo
EG Encar García-Oliver
MI María de la Iglesia-Vayá
SR Susana Rodríguez-Navarro
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Total RNA prepared by hot phenol extraction was treated for 30 min at 30 °C with DNase I RNase-free (Roche) prior to use for cDNA synthesis. Subsequently, cDNA was synthetized in 20 µL reactions containing 50 ng/µL of DNase I treated RNA, 250 ng of random hexamers (Invitrogen), 10 units/µL of SuperScript III Reverse Transcriptase (Invitrogen), 1× First Strand Buffer, 10 mM DTT, and 0.5 mM dNTPs, following the manufacturer’s instructions. Quantitative real-time PCR was then performed in a LightCycle 480 Thermal Cycler (Roche) using the SYBR® Premix Ex Taq™ kit (Takara) for fluorescent labeling. For each analysis primer pair, a negative control was included. The primers set used in this study is provided in Additional file 1: Table S3. A primer pair for ALG9 was used as a reference gene. Data and errors bars represent the average and standard deviation of three independent biological samples.

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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