Sybr green qpcr supermix udg with rox reference dye

The uppermost fully expanded leaves from at least three individual plants were harvested at 45 days after germination under natural long day (NLD) conditions. Total RNA was extracted from the pooled leaf materials using TRIzol Reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions and then treated with RNase-free DNase I (New England Biolabs, Hitchin, UK). First-strand cDNA was synthesised using M-MLV reverse transcriptase and oligo (dT) primer (Promega, Madison, WI, USA) as described by the manufacturer. Quantitative RT-PCR was carried out in a total volume of 10 μl, including 2 μl of cDNA template (5- to 10-fold dilutions), 0.5 μl of 10 μM gene-specific primer and 5 μl of SYBR Green qPCR Supermix-UDG with ROX Reference Dye (Invitrogen, Carlsbad, CA, USA). The qRT-PCR was conducted on a StepOne System (ABI) using the following parameters: 95°C for 10 min, followed by 40 cycles of 95 °C for 10 s and 60 °C for 30 s. The relative gene expression levels were calculated according to the ΔΔCT method described previously40 (link). The rice Actin1 gene was used as an internal control to normalise the data.

Total RNA was extracted with the TRIzol reagent from 1 × 10⁶ B16F10 cells or 100 mg of tissue sample following manufacturer instructions (Invitrogen). One microgram of total RNA was used for DNase treatment and subsequent reverse transcription using the ImProm-II protocol (Promega). For semiquantitative PCR, target genes were amplified using 100 ng of DNA and Taq platinum DNA polymerase (Invitrogen) following the manufacturer protocol. Samples were loaded in a 2.0% agarose gel stained with SYBR Safe (Invitrogen). For quantitative real-time PCR, we used 10–50 ng of complementary DNA (cDNA) and platinum SYBR Green qPCR SuperMix UDG with ROX reference dye (Invitrogen); results were analyzed using the ABI Prism 7000 sequence detection system. Transcripts were quantified relative to the housekeeping gene beta-actin using the Ct method (Livak and Schmittgen, 2001 (link)). The oligonucleotide primers used in the PCR analyses are listed in Tables S1 and S2.