create rna isolation and expression assay

assays/RNAIsolationAndExpressionAnalysis/protocols/RNAIsolationAndExpressionAnalysisProtocol.md

+## RNA isolation and expression analysis
+
+Total RNA was extracted from the roots of 18-day-old plants by standard phenol/chloroform extraction and LiCl precipitation. Thereafter, DNase treatment was performed and cDNA was synthesized from 600 ng of total RNA using the QuantiTect Reverse Transcription kit (QIAGEN) according to the manufacturer’s protocol. The product was diluted with autoclaved water to a final volume of 200 μl. Using quantitative real-time PCR (qPCR), 11 genes were examined using the gene-specific primers indicated in Supplementary Table S2. The qPCR was performed using the SYBR green as per the manufacturer’s instructions using a CFX96 Touch Real-Time PCR Detection System (Bio-Rad). All quantifications were normalized to the *TIP41* (AT4G34270) and *UBC21* (AT5G25760) genes using the 2−ΔΔCt method. The RT-PCRs were performed in duplicate for each of the four independent samples.
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