Quantitative RT-PCR Analysis of Auxin Signaling Genes in Arabidopsis

The qRT-PCR was performed as published previously with minor modifications.^{23 (link)} For RNA isolation, ~50 mg of At seedlings after were harvested with liquid nitrogen in the same light condition. Total RNA was isolated using Qiagen Plant RNeasy mini kit followed by cDNA preparation from 1 μg of RNA of each sample using Bio-Rad iScriptTM Reverse Transcription Super-mix. The quantitative RT-PCR (qRT-PCR) was performed using the CFX384 TouchTM Real-time detection system (Bio-Rad Laboratories), following the manual of iTaqTM universal SYBR green supermix. Gene-specific primers were designed using the Primer Quest tool. All reactions were carried out in Hard-shell 384-well PCR plates (provided by Bio-Rad, Cat #: HSP3805) Table 1. The PCR mix and thermocycler program for the qPCR were similar to those done by Singh, 2022.^{23 (link)} Transcripts level were normalized with ACTIN. Each qRT-PCR reaction was performed in three biological replicates and all data were presented as mean ± SEM.Table 1.

List of primers used in qRT-PCR.

GENES	Forward Primer	Reverse Primer
TIR1	TAATTTGGTACCTGACGGATGG	CACCATCCTCTTCAGCCTTATC
IAA14	CCTTCTAAGCCTCCTGCTAAAG	CTTCGCCGCTCTTCTGATTA
ARF7	CCCTCCAAGTTACTGAGCTTTC	GCTGAGGCAACTGAGACATT
LBD16	TCAAACCGGAGGAGGAGTAT	AGCCTGAAGCTCACCTAAATC
NAC1	GGATCTCATCATCCTCCCAATC	CAGCTTCCCATGTTGTCTCT
AIR3	GGATGACATTCCTGGACCTATC	GACCGGGATTCACAGCTAAA
ACTIN	CGATGAAGCTCAATCCAAACGA	CAGAGTCGAGCACAATACCG