Chapter 4 150 minimum three internodes were put in tube.. For qPCR we had three technical replicates. In the first experiment tomato plants were harvested after FR treatment after 1 hours, 2 hours and 6 hours. In the second experiment, the plant material was harvested 2 hours after pharmacological treatment. Samples were snap frozen by liquid nitrogen and stored at -80 degrees Celsius. Total RNA was extracted by the RNeasy kit (Qiagen) according to manufacturer’s instructions. RNA was quantified with nanodrop (Thermo Fisher Scientific). The reverse transcription was done using random hexamer primers with RevertAid H Minus (Thermo Fisher Scientific™) according to manufacturer’s instructions. 4.4.8 Quantitative RT-PCR The CFX opus 384, Bio-rad CFX Maestro was used to perform quantitative reverse transcription PCR. Primers used are listed in Table 4.3. We used SYBRgreen super mix dye (Thermo Fisher Scientific™). Relative transcript abundance was calculated using the comparative 2-ΔΔCt method using ACTIN2 as the reference gene. 4.4.9 Statistical analysis Data was analyzed by R v4.3 using an Anova followed by a Tukey test and Pearson correlation analysis. The illustration of the data was made by GraphPad (Version 9.5.1 (528), January 24, 2023).
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