Sybr premix kit

The quantitative real-time PCR was performed using the line-gene K FQD-48A PCR system with SYBR Premix Kit (Promega, WI, USA). Reactions were done in a total volume of 20 μL comprising 1 μL sense and antisense primer (2 μM), 10 μL SYBR Premix, 7 μL dH₂O, and 1 μL sample of genomic DNA. All primers used for quantitative PCR are listed in Table 2; PCR was performed at 95°C for 2 min, followed by 40 cycles of 95°C for 5 s and 60°C for 30 s; a melting curve was done at the end of the amplification for the specificity. Quantification was performed with the standard curve method using five standard dilutions, in triplicate, of two goats (BC186 and BC228) gDNA ranging from 16.50 to 0.33 ng (5000 to 100 haploid genome copies) per reaction. The copy number of hLA gene in each goat was estimated; ACTIN sequence was used as endogenous gene.

Total RNA was purified with TRIzol reagent (Invitrogen) according to the manufacturer's instructions. Two micrograms of total RNA were used for cDNA synthesis with a reverse transcription kit (Promega). Quantitative real-time PCR was performed using the SYBR premix kit (Promega), and the primer sequences used are listed in Supplemental Table S2. Gene expression was quantified by the comparative CT method and normalized to actin expression. Values are expressed as the mean ± SD. The experiments were repeated at least three times, and statistical analysis was performed on the individual experimental sets.

Total RNA was extracted from soybean and Arabidopsis plants using the RNAsolve reagent (OMEGA Bio-Tek, Norcross, GA, USA), and was then purified with RNase-free DNase I (Invitrogen, USA). About 1 μg of RNA was reversely transcribed to complementary DNA (cDNA) with MMLV-reverse transcriptase (Promega, Madison, WI, USA). qRT-PCR was performed using a SYBR Premix kit (Promega, Madison, WI, USA) on a Rotor-Gene 3000 real-time PCR system (Corbett Research, Mortlake, Australia). The GmEF-1a and AtEF-1a were separately used as an internal control. Relative expression of genes was calculated as the ratio of expression of the tested genes to those of EF-1a as described previously [56 (link)]. Specific gene primers are listed in Table S1.