480 real time pcr detection system

Reverse transcription of total RNA was performed using the PrimeScript^® RT reagent kit with gDNA Eraser (Takara, Dalian, China). qRT-PCR was performed on the Roche 480 real-time PCR detection system using LightCycler^® 480 SYBR Green I Master Mix (Roche Diagnostics, Indianapolis, IN, USA). The qRT-PCR reactions were performed under the following conditions: 5 min at 95 °C for the initial denaturation, followed by 42 cycles of 10 s at 95 °C, 20 s at 57 °C, and 20 s at 72 °C for the PCR amplification, and 1 cycle of 30 s at 95 °C, 30 s at 65 °C and 0.06 °C/s heating up to 95 °C for the melting curve. Data was analyzed using the 2^−ΔΔCT method as described by Livak & Schmittgen (2001) (link). All expression data obtained in the qRT-PCR assay were normalized to the expression of JcActin1 (Zhang et al., 2013 (link)) and AtActin2. The primers used for qRT-PCR are listed in Table S1.

Arabidopsis and Jatropha total RNAs were extracted from frozen tissue using TRIzol reagent (Transgene, China). Synthesis of the first-strand cDNA was performed following the methods described above. qRT-PCR was performed using SYBR^® Premix Ex Taq™ II (Takara Bio) on a Roche 480 Real-Time PCR Detection System (Roche Diagnostics).
The primers used for qRT-PCR are listed in lSupplementary Table S1. qRT-PCR was performed using two independent biological replicates and three technical replicates for each sample. Data were analysed using the 2^−ΔΔCT method as described by Livak and Schmittgen44 (link). The transcript levels of specific genes were normalised using Arabidopsis Actin2 or Jatropha Actin45 (link).

Total RNA was isolated from the aggregates using the total RNA Kit (Omega Bio-Tec, USA) following the manufacturer’s protocol. Total RNA (500 ng) was converted to cDNA using the reverse transcription kit (TaKaRa, JAPAN). All RT-PCRs were performed on a Roche 480 Real-Time PCR Detection System (Roche Diagnostics) in 20 μl reaction volume containing 10 μl of SYBR Green I Master Mix (Roche Diagnostics), 2 μl of 10 mM sense or antisense primer, and 6 μl of RNAse-free water. The expressions of the following genes were examined: type X collagen (COL10A1), alkaline phosphatase (ALP), runt-related transcription factor 2 (RUNX2), Indian hedgehog (IHH), parathyroid hormone-related protein receptor (PTHrP-R), and parathyroid hormone-related protein (PTHrP) mRNAs. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA was used for normalization of gene expression. The PCR reaction conditions were as follows: 1 min at 95 °C, followed by 39 amplification cycles of 15 s at 95 °C, 15 s at 60 °C, and 20 s at 72 °C. After the last cycle, a melt curve was generated. The Ct value of GAPDH mRNA was subtracted from the Ct value of the gene of interest (ΔCt). The average ΔCt value of the triplicate reactions was determined. MSCs cultured in the pellet culture system were used as controls. The relative expression level for each gene was expressed as fold change by the 2^−ΔΔCT method.

Jatropha total RNA was extracted from frozen tissue as described by Ding et al. [48 (link)] Arabidopsis total RNA was extracted from frozen tissue using TRIzol reagent (Transgene, China). First-strand cDNA was synthesized using the PrimeScript® RT Reagent Kit with gDNA Eraser (TAKARA, Dalian, China) according to the manufacturer’s instructions. qRT-PCR was performed using SYBR® Premix Ex Taq™ II (TAKARA) on a Roche 480 Real-Time PCR Detection System (Roche Diagnostics).
The primes used for qRT-PCR are listed in Table S1. qRT-PCR was performed using two independent biological replicates and three technical replicates for each sample. Data were analyzed using the 2^–ΔΔCT method as described by Livak and Schmittgen [50 (link)]. The transcript levels of specific genes were normalized using Jatropha Actin1 or Arabidopsis Actin2.

Total RNA was extracted from each tissue, and first-strand cDNA was synthesized with a PrimeScript^® RT Reagent Kit with gDNA Eraser (Takara, Dalian, China) according to the manufacturer’s instructions. qRT-PCR was performed with LightCycler^® 480 SYBR Green I Master (Roche, Indianapolis, IN, USA) on the Roche 480 Real-Time PCR Detection System (Roche Diagnostics, Mannheim, Germany). qRT-PCR was performed with two independent biological replicates (tissue samples were harvested from different plants) and three technical replicates for each sample. Data were analyzed using the 2^−ΔΔCT method as described by Livak & Schmittgen (2001) (link). Expression levels of specific genes were normalized to that of the actin gene in Jatropha (Zhang et al., 2013 (link)). Primers used in qRT-PCR are listed in Table S3.

The roots, stems, mature leaves, inflorescence buds, flower buds, male flowers, female flowers and fruits of mature Jatropha plants and the aboveground tissues of 15 days Arabidopsis seedlings were collected for qRT-RCR detection. Total RNA was extracted from frozen Jatropha tissues as described by Ding et al. (2008) (link). Total RNA was extracted from frozen Arabidopsis tissues using TRIzol reagent (Transgene, China). First-strand cDNA was synthesized with the PrimeScript^® RT Reagent Kit with gDNA Eraser (TAKARA, Dalian, China). The cDNA templates of first-strand cDNA were diluted 5-fold with sterilized double-distilled water. qRT-PCR was performed using SYBR^® Premix Ex Taq™ II (TAKARA, Dalian, China) on a Roche 480 Real-Time PCR Detection System (Roche, Mannheim, Germany). The primers employed for qRT-PCR are listed in Table S1. qRT-PCR was conducted with three independent biological replicates and three technical replicates for each sample. The data were analyzed using the 2 ${}^{-\Delta \Delta \mathrm{CT}}$ method described by Livak & Schmittgen (2001) (link). The transcript levels of specific genes were normalized using Jatropha ACTIN1 or Arabidopsis ACTIN2.