Superscript 3 first strand synthesis system rt pcr kit

Total RNA was isolated from Hca-F cells using the TRIzol reagent (Invitrogen, Carlsbad, CA, USA), according to the manufacturer’s instructions. The concentration and purity of RNA were determined at 260/280 nm using a Nanodrop spectrophotometer (Thermo Scientific, Rockford, IL, USA). Total RNA (1.0 µg) was then reverse-transcribed using the SuperScript III first-strand synthesis system RT-PCR kit (Invitrogen, Carlsbad, CA, USA), according to the manufacturer’s protocol. Additionally, the target cDNAs were amplified using primer pairs for hif-1α, vegfr-3, vegf-c, hgf and c-met. Gapdh was used as the internal standard. The sequences of the forward and reverse primers and the product lengths are shown in Table 1. All primers were synthesized by Takara (Dalian, China). The relative quantitation of mRNA expression levels was determined using the relative standard curve method according to the manufacturer’s instructions.

For the amplification of full length cDNA of TaSERKs, RNA was isolated from the wheat embryogenic calli and cDNA prepared by one step RT–PCR using Superscript® III First–Strand Synthesis System RT–PCR kit (Invitrogen, USA) was used as a template for the amplification of TaSERK genes using Phusion High Fidelity Taq polymerase (Finnzymes). The thermal cycling condition was as follows: initial denaturation at 98 °C for 30 s followed by amplification for 35 cycles at 98 °C for 10 s, annealing at 62 °C for 30 s, extension at 72 °C for 1 min and a final extension at 72 °C for 7 min. Each of the full length amplified products of all TaSERKs obtained were then cloned individually in pDRIVE vector (PCR cloning kit, Qiagen, Germany) and entry vector pENTR^TM/D–TOPO (Invitrogen Inc. USA) as described previously^{39 (link)}. Primers used for above cloning were listed in Supplementary Table S2.

RNA was extracted from cell lines as described previously, according to the manufacturer’s instructions [48 (link)]. Cells were lysed in a 6 well plate using TRIzol reagent (Invitrogen), and total RNA resuspended in RNase-free water was quantified using a Nanodrop 2000 spectrophotometer (ThermoFisher Scientific, Waltham, MA, USA). To generate cDNA for qtRT -PCR, 1 mg RNA was used with the SuperScript III First-Strand Synthesis System RT-PCR kit (Invitrogen, Waltham, MA, USA). qtRT -PCR was done as described previously, using the C1000 Touch Thermal Cycler CFX96 Real-Time System (Bio-Rad, Hercules, CA, USA). Oligonucleotide primers used for qtRT-PCR were described in [49 (link)].