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qPCR and validation of RNA-seq data

KG Kelsey D. Galimba
DB Daniel G. Bullock
CD Chris Dardick
ZL Zhongchi Liu
AC Ann M. Callahan
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Quantitative real-time PCR on total RNA was conducted with an ABI PRISM® 7900 Sequence Detection System (Thermo Fisher Scientific, MA) on five genes, to validate transcript per million (TPM) values from the transcriptome. Amplifications were completed with Invitrogen Superscript III Platinum SYBR Green One-Step qPCR kit with ROX (Thermo Fisher Scientific, MA). Primers (Figure S1) were validated prior to use, to confirm single product amplification and consistent efficiencies through dilution curves. RNA samples were run in triplicate and normalized to Apple TRANSLATION ELOGATION FACTOR 2 (MdTEF2) using the 2∆∆Ct method43. All values were standardized to GA3-treated hypanthium and compared with both identically-standardized TPM values from RNA-seq, and TPM values that had first been standardized to MdTEF2 (Figure S5).

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