Cfx 96 real time pcr system

Total RNAs of A-E stage leaves, stems, roots, flowers, and mature leaves under 200 mM NaCl treatment for 24 h were extracted for real-time PCR. qPCR primers were designed using Beacon Designer™ Free Edition software (version 7.8). The amplification procedure consisted of 94°C for 5 min, followed by 35 cycles of 30 s at 94°C, 1 min at 51°C, and 1 min at 72°C. SYBR Green qPCR was carried out in a fluorometric thermal cycler (Bio-Rad CFX96^™ Realtime PCR System) using SYBR Premix Ex Taq^™ (TaKaRa, Mountain View, CA) in 20 µl reaction mixtures containing 10 µl of 2×SYBR Premix Ex Taq™ (TaKaRa), 50 ng cDNA, and 0.2 µM each primer (Table S1, Lbtubulin RT and LbTTG1 RT) using Lbtubulin as an internal control. The PCR thermal cycle was as follows: denaturation at 95°C for 5 min and 40 cycles at 94°C for 20 s, 58°C for 15 s, and 65°C for 15 s. Three replicate biological experiments were performed. Relative expression levels were calculated using the formula 2^–ΔΔC(T).

The RNA samples were treated with RQ1 RNase-Free DNase (Promega, USA) to eliminate residual genomic DNA and reverse transcribed using the PrimeScript® RT Reagent Kit (TaKaRa, Japan), and the qRT-PCR reactions were performed on a Bio-Rad CFX96 Real-Time PCR System using the SYBR Green EX Taq mix (TaKaRa, Japan) with the kit-recommended two-step standard cycling method. Every qRT-PCR was performed in triplicate with a volume of 25 μL and repeated more than three times under the same conditions in a separate experiment. For the relative quantification of specific genes from different transcripts, the cycle threshold (C_T) method was used to calculate fold changes in expression. The 16S rRNA gene was chosen as the reference gene to normalise the variability in expression levels. The orf_1053, orf_1589, orf_2472 and orf_2475 genes were selected to verify the RNA sequencing results. Primer Premier 5 was used to design the forward and reverse primers for each selected gene. The efficiency of each primer pair was calculated using the standard curves of 10-fold serial dilutions of genomic DNA by the Bio-Rad CFX Manager.

The tissues and cells were extracted to RNA and then reversed to cDNA for conducting qRT-PCR assay by TRIzol reagent and reverse transcription kit (Invitrogen). Actin and U6 were used as reference genes for mRNA and miRNA, respectively. qRT-PCR analysis was evaluated by a CFX96 real-time PCR system with SYBR (TaKaRa, China). The relative expressions were calculated by using 2^−ΔΔCt method. The primers are listed in Table 2.Table 2.

The list of primers

Name	Forward/reverse	Sequence (5ʹ to 3ʹ)
CASC7	F	AACATGGTCTCTTGGTGCCTGATG
	R	CCACGGTAAGCGACGAGGAATC
miR-21-5p	F	GCTTATCAGACTGATGTTG
	R	GAACATGTCTGCGTATCTC
FASLG	F	TGCCTTGGTAGGATTGGGC
	R	GCTGGTAGACTCTCGGAGTTC
actin	F	CATGTACGTTGCTATCCAGGC
	R	CTCCTTAATGTCACGCACGAT
U6	F	CTCGCTTCGGCAGCACA
	R	AACGCTTCACGAATTTGCGT