Abi 7300 real time detection system

Total RNA extraction of plant samples was performed using the Eastep^®Super Total RNA Extraction Kit (Qiagen, Promega, Shanghai, China). First-strand cDNA was synthesized using the ReverTra Ace qPCR RT Master Mix with the gDNA Remover Kit (Toyobo, Osaka, Japan). Real-time RT-PCR was performed using the ABI7300 Real-time Detection System (Applied Biosystems, Foster City, CA, USA) with the SYBR Premix Ex TaqTM II (Toyobo, Shanghai, China). The AtUBC9 and BnaEF1-a genes were used as reference genes. The relative expression levels of the target genes were calculated by the 2^−∆∆CT method [51 (link)]. All the primers used in this study are listed in Table S1.

Total RNA was isolated using the total RNA kit (Tiangen, Beijing, China) and then reverse transcribed into cDNA using the PrimeScript RT reagent Kit (Toyobo, Shanghai, China). qRT–PCR was performed using ABI 7300 Real-time Detection System (Applied Biosystems, USA) with SYBR Premix Ex TaqTM II (Toyobo, Shanghai, China). The PCR conditions were set as follows: 95°C for 2 min; 40 cycles of 95°C for 30 s and 55°C for 30 s; and 72°C for 1 min, and the experiments were repeated three biological replicates. The ΔΔC_T method was used to calculate relative expression levels of MtERF genes using GAPDH as reference gene. Primers of nine MtERF genes (randomly selected from DREB subfamily) and GAPDH gene used for qRT-PCR detection are listed in Table S1.

Total RNA was extracted from fish tissues and used for cDNA synthesis as described previously (Zhang et al. 2008a (link)). qRT-PCR was performed in an ABI 7300 Real-time Detection System (Applied Biosystems, Foster City, CA, USA) using the SYBR ExScript qRT-PCR Kit (Takara, Dalian, China) as described previously (Zhang et al. 2008a (link)). Each assay was performed in triplicate with 16S rRNA as control. All data are given in terms of relative mRNA expressed as means plus or minus standard errors of the means (SE).

Total RNA extraction of plant samples was performed using the Eastep^®Super Total RNA Extraction Kit (Qiagen, Promega, Shanghai, China). First-strand cDNA was synthesized using the ReverTra Ace qPCR RT Master Mix with the gDNA Remover Kit (Toyobo, Osaka, Japan). Real-time RT-PCR was performed using the ABI7300 Real-time Detection System (Applied Biosystems, Foster City, CA, USA) with the SYBR Premix Ex TaqTM II (Toyobo, Shanghai, China). The AtUBC9 and BnaEF1-a genes were used as reference genes. The relative expression levels of the target genes were calculated by the 2^−∆∆CT method [51 (link)]. All the primers used in this study are listed in Table S1.

qPCR was performed on an ABI 7300 Real-time Detection System (Applied Biosystems). The standard curve of 10¹² − 10⁷ was drawn according to the concentrations of plasmid DNA. Reaction were carried out in a total volume of 10 μl, containing 1 μl of diluted cDNA mix, 1 μl of each primer (10 mM), 5 μl of iTaq Supermix (Bio-Rad, USA), and 2 μl of Milli-Q water. The reaction procedures was as follows, 94 °C for 3 min, followed 39 cycles with each cycle at 94 °C for 10 s, 53 °C for 20 s, and 72 °C for 30 s. Melting curves were monitored and recorded during each change of temperature, rising from 65 to 95 °C, with three repetitions per sample. To confirm that only one PCR product was amplified and detected, dissociation curve analysis of amplification products was performed at the end of each PCR reaction. After the PCR program, data were analyzed with ABI 7300 SDS software (Applied Biosystems). The comparative CT method was used to analyze the expression levels of the gene.

The total RNA was extracted from the tumor tissues and lung cancer cells using TRIzol reagent (Invitrogen, Carlsbad, CA). Complementary DNA was synthesized with murine leukemia virus‐reverse transcriptase (Invitrogen), and each reaction was performed in triplicate using SYBR Green (Tiangen, Beijing, China) according to the manufacturer's instructions using an ABI7300 real‐time detection system (Applied Biosystems). Glyceraldehyde 3‐phosphate dehydrogenase was used as an internal control. The program was as follows: 95°C for 2 minutes and 40 cycles of 95°C for 15 seconds, 60°C for 30 seconds and 68°C for 30 seconds. A dissociation curve was generated. The fold change in expression was calculated by the formula 2−ΔΔCt. The primers used in the experiments are listed in Table S1. The quantitative real‐time polymerase chain reaction (qRT‐PCR) data were calculated from triplicate reactions.

Total RNA was isolated using the total RNA kit (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. First-strand cDNA was synthesized using the Prime Script RT reagent Kit (Promega, Madison, WI, USA). Real-time RT-PCR was performed using ABI7300 Real-time Detection System (Applied Biosystems, Foster City, CA, USA) with SYBR Premix Ex TaqTM II (Toyobo, Shanghai, China). The PCR conditions were set as follows: 95 °C for 2 min; 40 cycles of 95 °C for 30 s and 60 °C for 30 s; and 72 °C for 1 min. The 2^−∆∆CT method was used to calculate the relative expression levels of BnaCRF8 genes using Actin as the reference gene. The primers of the 4 BnaCRF8 genes and the Actin gene used for qRT-PCR detection are listed in Table S2.