Sybr green pcr master mix kit

Young leaves of A. annua plants were used for RNA isolation by plant RNA Kit (Tiangen) according to the manufacturer’s instructions. The isolated total RNA was reverse-transcribed into cDNA using the TaKaRa cDNA synthesis kit. qRT-PCR assays using SYBR Green PCR Master Mix kit (Tiangen) as a detector were performed in Roch LightCycler^®96 system according to the manufacturer’s instructions. The comparative cycle threshold (CT) method was estimated relative transcript levels of samples and means of triplicate independent PCRs. The β-actin gene of A. annua was used to estimate relative mRNA levels. The qRT-PCR primers are listed in Supplementary Table S1. The process for amplification was 5 min at 95°C, followed by 40 cycles of 95°C for 10 s, and 40 s at 60°C for primer annealing and elongation. Then a dissociation stage for 20 s at 95°C, 40 s at 60°C, and 20 s at 95°C.
The 2^-ΔΔCT method was used for estimating the relative gene expression level according to the following formula (Livak and Schmittgen, 2001 (link)):

Cellular RNA was extracted with TRIZOL Reagent (Invitrogen, USA). cDNA was synthesized using Reverse Transcriptase (TianGen Biotech, China). Real-time PCR was performed using the SYBR-green PCR master mix kit (TianGen Biotech, China). The data were analyzed by using Rotor-gene Q software (ver. 1.7). Relative mRNA levels were calculated by the 2^-DDCt method (n = 6). All primers used in this study were listed in Table 1.Table 1

Primers used for real-time PCR

Gene	Primer	Sequence (5’–3’)
PPARγ	sense	GCAGCTACTGCATGTGATCAAGA
	Anti-sense	GTCAGCGGGTGGGACTTTC
ABCA1	sense	CGTTTCCGGGAAGTGTCCTA
	Anti-sense	GCTAGAGATGACAAGGAGGATGGA
ABCG1	sense	GGGAAGTTGATAAAGGATGT
	Anti-sense	GATTCGGGCTATGTATGG
SR-BI	sense	ATCTGGTGGACAAATGGAA
	Anti-sense	GAAGCGATACGTGGGAAT
apoA-I	sense	GGCACGTATGGCAGCAAGAT
	Anti-sense	CCCAGAAGTCCCGAGTCAAT
ABCG5	sense	AGCGTCAGCAACCGTGTC
	Anti-sense	AGCAGCGTGGTCTTCCCT
ABCG8	sense	TTAAGCCACTCCCAATACA
	Anti-sense	GTTGCTCCAAGAATAAATGA
ABCB11	sense	CAAATAAGGTTGTGGGTAA
	Anti-sense	AGGACTGACAGCGAGAAT
ABCB4	sense	CCCCACAGAGGGTAAGAT
	Anti-sense	CCAACCAGGGTGTCAAAT
LXRα	sense	TTTGAGCAGCGTCCATTC
	Anti-sense	GCAGTCAGTGAGCCTTCG
HMGR	sense	TGTTCACGCTCATAGTCGC
	Anti-sense	CTCCGCTGTGCTGTTCTG
LDLR	sense	GCCCAAGTCGCCATTCTC
	Anti-sense	GCCTGAGGTCCCATCCAA
CYP7A1	sense	TGGGCATCTCAAGCAAACAC
	Anti-sense	TCATTGCTTCAGGGCTCCTG

The total RNA was isolated using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s protocol. First-strand cDNA was generated using a Reverse Transcription System (Vazyme, Nanjing, China) following the manufacturer’s instructions. A real-time quantitative PCR (qPCR) experiment was performed using the SYBR Green PCR Master Mix kit (Tiangen Biotech, Beijing, China) and the IQ5 light cycler system (Bio-Rad, Hercules, CA, USA). The parameters for qPCR have previously been described [41 (link)]. The wheat β-actin and AtActin2 genes were used as internal controls in wheat and Arabidopsis, respectively. The samples were assessed using three biological replicates. The relative gene expression was calculated using the comparative CT method [42 (link)].