Rotor gene 3000 real time pcr detection system

To validate the RNA-Seq results, seven genes were selected randomly from shoots and then analyzed using quantitative real-time PCR (qRT-PCR). After rice seedlings were treated with or without 75 μM CdCl₂ solutions of 7 d, the shoots samples were harvested for RNA extraction. Gene-specific primer pairs were designed using Primer 5.0 software (Premier Biosoft International) as shown in Table S1 and plant samples and total RNA isolation were carried out as described above. ACTIN was used as an internal standard⁴³ . Two micrograms RNA was reverse-transcribed into cDNA using the iScriptTM advanced cDNA Synthesis Kit (Promega, WI, USA) after treated with RNase-free DNase I (Promega, Madison, WI), and the standard curve of each gene was prepared with several dilutions of cDNA. Quantitative real time PCR was performed using a Rotor-Gene 3000 real-time PCR detection system (Qiagen) with SYBR® qPCR Mix (Toyobo, Tokyo, Japan). Quantitative PCR reactions cycling conditions were performed as follows: 95 °C for 2 min, followed by 40 cycles at 95 °C for 15 s, 60 °C for 15 s, and 72 °C for 30 s. The relative expression value of the different genes was calculated using 2^−ΔΔCt method⁴⁴ . The experiment was performed three biological replicates.

Total RNA was isolated from the leaf, stem, bulbs, and roots of lily plants subjected to 4°C cold treatments, as described above. First-strand cDNA synthesis was performed using Superscript II reverse transcriptase (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions, using 1 μg total RNA and oligo(dT) primers. qRT-PCR was performed using a Rotor-Gene 3000 real-time PCR detection system (Qiagen) using SYBR® qPCR Mix (Toyobo, Tokyo, Japan) according to the manufacturer’s protocol. The primers used in this study were designed with Beacon Designer (Premier, Palo Alto, CA, USA) and are listed in Table 1. Real-time PCRs was carried out using prepared cDNA (80 μg) with each set of primers and probe and iQ™ SYBR® Green Supermix (Cat. No.170-8882, Bio-Rad, Hercules, CA, USA). The PCR cycling conditions were as follows:95°C (30 s), 60°C (30 s), and 72°C (15 s). All reactions were performed in biological triplicates. Relative mRNA levels were calculated using the 2^-△△Ct method [44 (link)] against the internal reference gene TIP1, with expression in CT 0 h used as the internal control. The sequences of primers used for QRT-PCR are listed in Table 4.

Samples of wound beds and subcutaneous implants were quickly excised, immediately frozen in liquid nitrogen, and stored at −80°C. Total RNA was extracted from 50 mg frozen tissue using a RNeasy Lipid Tissue Mini Kit (Qiagen, Hilden, Germany), according to the manufacturer’s instructions. cDNA was amplified in 40 cycles using the QuantiTect Reverse Transcription Kit (Qiagen, Germany) and Rotor-Gene 3,000 Real-Time PCR Detection System (Corbett Research, Sydney, Australia). GAPDH was used as the reference gene. The primer sequences were as follows: peroxisome proliferator-activated receptor gamma (PPARG), forward 5′-TCG​CTG​ATG​CAC​TGC​CTA​TG-3′ and reverse 5′-GAG AGG​TCC​ACA​GAG​CTG​ATT-3′; CCAAT-enhancer-binding protein alpha (CEBPA), forward, 5′-CTT​GAT​GCA​ATC​CGG​ATC​AAA​C-3′ and reverse, 5′-CCC​GCA​GGA​ACA​TCT​TTA​AGT-3′; vascular endothelial growth factor A (VEGFA), forward 5′-TTA​CTG​CTG​TAC​CTC​CAC​C-3′ and reverse 5′-ACA​GGA​CGG​CTT​GAA​GAT​G-3′; basic fibroblast growth factor (bFGF), forward 5′- AGC​GGC​TGT​ACT​GCA​AAA​ACG​G-3′ and reverse 5′-CCT​TTG​ATA​GAC​ACA​ACT​CCT​CTC-3’. Expression levels were calculated using the 2^−ΔΔCT method.

RNA was extracted by using Qiagen RNeasy Mini kit according to manufacturer instructions. Total RNA (3 mg in 25 ml) was reversely transcribed and produced cDNA that was used to detect the transcripts using the Rotor-Gene 3000 Real-Time PCR Detection System (Corbett Research, Sydney, NSW, Australia) with SYBR® Premix Ex Taq™. The expression level of GADPH housekeeping gene was used for normalization of SET mRNA expression level. Forward primer: 5’-CTTCAACTCTGGTCAAATAATGCA-3’, reverse primer: 5’-GAACAAAAATATAACAAACTCCGC-3’, forward primer: 5′-GAAGGTGAAGGTCGGAGTC-3′ and reverse primer: 5′-GAAGATGGTGATGGGATTTC-3′ for GADPH.

Adipose tissue was collected, frozen in liquid nitrogen, and stored at −80°C. RNA was extracted from 50 mg tissue samples using RNeasy Lipid Tissue Mini Kits (Qiagen, Hilden, Germany), according to the manufacturer’s instructions. cDNA was synthesized and amplified over 40 cycles using a QuantiTect Reverse Transcription Kit (Qiagen) and a Rotor-Gene 3000 Real-Time PCR Detection System (Corbett Research, Sydney, Australia). Expression levels were calculated using the 2−ΔΔCt method. The primer sequences were as follows: Ucp1: forward 5′-CTG​ATG​AAG​TCC​AGA​CAG​ACA​G-3′ and reverse 5′-CCA​GCA​TAG​AAG​CCC​AAT​GA-3′; PRDM16: forward 5′-CAG​CAC​GGT​GAA​GCC​ATT​C-3′ and reverse 5′-GCG​TGC​ATC​CGC​TTG​TG-3′; PGC1-α: forward 5′-CGA​CAG​CTA​TGA​AGC​CTA​TGA​G-3′ and reverse 5′-CTT​CTG​CCT​CTC​TCT​CTG​TTT​G-3′; FGF21: forward: 5′-CTG​GGG​GTC​TAC​CAA​GCA​TA-3′ and reverse 5′-CAC​CCA​GGA​TTT​GAA​TGA​CC-3′; and VEGF-A: forward 5′-GGA​GAT​CCT​TCG​AGG​AGC​ACT​T-3′ and reverse 5′-GGCGATTTAGCAGCAGATATAAGAA-3′HIF1α: forward: 5′-CAA​GAT​CTC​GGC​GAA​GCA​A-3′ and reverse 5′-GGT​GAG​CCT​CAT​AAC​AGA​AGC​TTT-3′.