Takara extaq r pcr kit

qRT-PCR was performed as described [21 (link)], using the TaKaRa ExTaq R PCR Kit, SYBR green dye (TaKaRa, Dalian, China) and a DNA Engine Opticon 2 machine (MJ Research, Waltham, MA). Fifteen genes including cellulose and lignin biosynthesis genes (Pt-CESA2.1, Pt-ATH.2, Pt-GLAC90.1, Pt-PRX1.8, Pt-PAL1.2 and Pt-PAL1.3) were validated, and the primers are shown in Additional file 1. The efficiency of the primers was calculated by performing real-time PCR on several dilutions of first-strand cDNAs. Efficiencies of the different primer sets were similar. The specificity of each primer set was checked by sequencing PCR products. The reactions were carried out in a 20 μl volume containing 2 μl of diluted cDNA, 200 nM of each primer, and PCR Master Mix with the following conditions: 95°C for 30 s, and 45 cycles of 95°C for 5 s, 58°C for 15 s, and 72°C for 20 s. Then, a thermal denaturing cycle of 95°C for 15 s and 60°C for 1 min was applied to determine the dissociation curves, which were used to verify the specificity of PCR amplifications. All reactions were run in triplicate for each sample. Relative expression levels of candidate genes were calculated by the 2^−ΔCt method. The results obtained for the different tissues were analyzed and standardized to the mRNA levels of poplar ACTINII-like (Accession number: EF145577), which shows stable expression.

Quantitative PCR (qPCR) was performed using the TaKaRa ExTaq R PCR Kit, SYBR green dye (TaKaRa, Dalian, China) and a DNA Engine Opticon 2 machine (MJ Research). The qPCR program included an initial denaturation at 94°C for 5 min, followed by 40 cycles of 30 s at 94°C, 30 s at 58°C, and 30 s at 72°C, and a final melt-curve 70–95°C. The melting curve was used to check the specificity of the amplified fragment. All reactions were carried out in triplicate for technical and biological repetitions of three individuals. The generated real-time data were analyzed using the Opticon Monitor Analysis Software 3.1 tool. Specific primer sets were designed to target the 3′ untranslated region (UTR) of each gene using Primer Express 3.0 software (Applied Biosystems). The real-time PCR primer pairs are shown in Additional file 1. The efficiency of the primer sets was calculated by performing real-time PCR on several dilutions of first-strand cDNAs. Efficiencies of the different primer sets were similar. The specificity of each primer set was checked by sequencing PCR products [34 (link)]. The results obtained for the different tissues analyzed were standardized to the transcript levels for PtACTIN (Additional file 2).