Iq5 rt pcr instrument

The rubber tree DNA and RNA isolation was carried out as described previously [34 (link)]. The Arabidopsis total RNA was extracted using Qiagen RNeasy Plant Mini Kit (Qiagen, USA). The RNA samples used for RT-PCR analysis were treated with DNase I to remove possible DNA contamination. Two microgram total RNA was used for the each detection. The first-strand cDNA was synthesized with SuperScript III (Invitrogen) according the instructions. Quantitative RT-PCR was carried out using IQ SYBR green supermix (Bio-Rad, USA) with gene specific primers listed in S1 Table. PCR was performed on a Bio-Rad Iq5 RT PCR instrument using following program: 95°C for 3 min, followed by 40 cycles of 95°C for 15 sec, 60°C for 30 sec and 72°C for 30 sec.
For Northern blotting, HbCBF1 probe was prepared from full length cDNA with DIG Northern Starter Kit (Roche, USA). Twenty micrograms of total RNA was run on 1.5% denature agarose gels and the blot was then transferred onto Amersham Hybond^TM-N⁺ nylon membranes (GE Healthcare, USA). The hybridization and detection was carried out according to the manufacturer’s instructions.

Total RNA was extracted from different tissues of grape hyacinth, including roots, bulbs, and leaves, and from the flowers at five developmental stages using the Total RNA Kit (Omega, Norcross, GA, USA). The five stages (S1–S5) of petals of grape hyacinth were divided according to the petal pigmentation [30 (link)] (Figure 2A). Next, 1 μg of RNA was used to synthesize cDNA by using the PrimeScript™ RT reagent Kit with gDNA Eraser (Takara Biotechnology, Dalian, China), following the manufacturer’s instructions. Then, qRT-PCR was carried out on the iQ5 RT-PCR instrument (Bio-Rad, Hercules, CA, USA) using AceQqPCR SYBR^® Green Master Mix (Vazyme, Nanjing, China). The MaActin gene was chosen as the internal control gene for Muscari. Spp, and the NtTubA1 gene was used as the internal control gene for tabacum samples. Primers used for qRT-PCR are listed in Table S1. The relative expression of MaDFR was quantified using the 2^−△△Ct method [31 (link)]. Using the Correlationplot Rectanglel module of Qi MetA software (Qiji Biotechnology Co, Ltd, Beijing, China), the r value of MaDFR expression and anthocyanin accumulation of the five stages of flower development were recalculated using the Pearson method, not including the roots, bulbs, and leaves. All analyses were conducted with three independent experiments.

Isolation of rubber tree DNA and RNA was carried out as described (Zewei An, 2012 (link)). For northern blotting, a HbEBP1 probe was prepared from full-length cDNA with DIG Northern Starter Kit (Roche, USA). Twenty micrograms of total RNA was run on a denature 1.5% agarose gel and the blot was then transferred onto an Amersham Hybond^TM-N⁺ nylon membrane (GE Healthcare, USA). Hybridization and detection were carried out according to the manufacturer’s instructions.
Total RNA was extracted from Arabidopsis seedlings using Qiagen RNeasy Plant Mini Kit (Qiagen, USA). For quantitative RT-PCR (Q-PCR) analysis, the RNA samples were treated with DNase I to remove possible DNA contamination. Two micrograms of total RNA was used for each detection. The first-strand cDNA was synthesized with SuperScript III according to the manufacturer’s instructions (Invitrogen, USA). Q-PCR was carried out using IQ SYBR Green Supermix (Bio-Rad, USA) with gene-specific primers as listed in Supplemental Table S1. PCR was performed on a Bio-Rad Iq5 RT PCR instrument using the following program: 95°C for 3 min, followed by 40 cycles of 95°C for 15 s, 60°C for 30 s and 72°C for 30 s.