Iq5 rt pcr detection system

Trizol reagent (Takara, Dalian, China) was used for isolating total RNA from ventricular tissue. 50–100 mg of tissue was directly lysed by mixing with 1 mL of Trizol reagent and homogenized using a homogenizer. Then 0.2 mL of chloroform was added to the homogenized sample, and incubated for 20 min at room temperature. Subsequently, RNA was precipitated by mixing with isopropyl alcohol. Total RNA yield was quantified by microplate reader (Molecular Devices, Sunnyvale, CA, USA) measured at 260 nm. Then mRNA was isolated from total RNA by using Oligo (dT), and reverse transcribed into first-strand complement DNA (cDNA) and amplified using a PrimeScript 1st strand cDNA synthesis kit (Takara). Reaction system included 2 μL of cDNA, 12.5 μL of 2 × SYBR Green 1 Master Mix (Takara), and 1 μL of each primer. The PCR condition was as follows: pre-incubation at 95 °C for 30 s, followed by 40 cycles of denaturation at 95 °C for 5 sec, and annealing/extension at 60 °C for 30 s using iQ5 RT-PCR detection system (Bio-Rad, Hercules, CA, USA). The primers were listed as below:
CD31 Forward 5′-TATCCAAGGTCAGCAGCATCGTGG-3′, Reverse 5′-GGGTTGTCTTTGAATACCGCAG-3′
VEGF Forward 5′-CTGTGTGCCCCTGATGCGATGC-3′, Reverse 5′-CCTCCGGACCCAAAGTGCTCTG-3′

Total RNA was extracted from frozen tissue of the flowers, roots, bulbs, and leaves of grape hyacinth (M. aucheri “White Beauty” and M. aucheri “Dark eyes”) as well as flowers of tobacco (NC89), using the Omega Total RNA Kit (Omega, Norcross, GA, USA). Purified RNA was assessed using agarose electrophoresis and measured on a Nanodrop 2000 (Thermo Scientific). Then, 1-μg RNA aliquots were used for reverse transcription to cDNA, using the PrimeScript™ RT Reagent Kit (TaKaRa Biotechnology, Dalian, China). The cDNA was diluted five-fold and used as the template for qRT-PCR. NovoStart^®SYBR qPCR SuperMix Plus (Novoprotein, Shanghai, China) was used as the fluorochrome for the qRT-PCR assay. The assay was conducted using the iQ5 RT-PCR detection system (Bio-Rad, Hercules, CA, USA). Each reaction mixture consisted of NovoStart^®SYBR qPCR SuperMix Plus with 0.8 μL of forward and reverse primers each, 1 μL of cDNA, and 7.4 μL of ddH₂O in a final volume of 20 μL. The amplification protocol was 95 °C for 1 min, followed by 40 cycles of 95 °C for 20 s, 58 to 62 °C for 20 s, and 72 °C for 30 s. The qRT-PCR primers of grape hyacinth and tobacco are listed in Table S1. MaActin and NtTubA1 were used as the internal control genes in each grape hyacinth and tobacco sample, respectively. All analyses were conducted in technical triplicate.

Small RNA was isolated using PureLink MicroRNA Isolation Kit (Invitrogen) and treated with TURBO DNA-free DNase (Ambion). Poly(A) tailing and cDNA synthesis of the DNAse-treated small RNA were performed using Ncode VILO MicroRNA cDNA Synthesis Kit (Invitrogen) according to the manufacturer's protocol. The forward primers for qRT-PCR analysis were designed based on entire known mature microRNA sequence, with additional 3 “A”s at the 3′ end to improve amplification specificity (Table 2). The reverse primer used was the Universal Primer in the EXPRESS SYBR GreenER MicroRNA qRT-PCR Kit (Invitrogen). 5S rRNA was selected as the internal reference gene for PCR quantification. To determine absolute copy number, a standard curve was generated using a synthetic LIN-4 RNA oligonucleotide.
qPCR was performed on iQ5 RT-PCR Detection System (BioRad). All reactions were run in triplicate.

Total RNA was isolated using the PureLink RNA Mini Kit (Thermo Fisher Scientific) and treated with DNase I (Thermo Fisher Scientific) according to the manufacturer’s instructions. Reverse transcription was carried out employing the Maxima H Minus DNA Synthesis Kit (ThermoFisher Scientific). The SYBR Select Master Mix (ThermoFisher Scientific) was used for qRT-PCR, following the manufacturer’s instructions. Reactions were conducted with the IQ5 rtPCR Detection System (BioRad, Munich, Germany). Transcripts of target genes were amplified using the gene-specific primers for human CFTR 5′-GGGCTGTGTCCTAAGCCATGGCCA-3′ and 5′-GATGGCTTGCCGGAAGAGGCTCC-3′. Absolute quantification was performed using a several fold dilution of target specific plasmid-DNA as internal standard curve. The resulting molecule concentrations were normalized to a reference gene (Mrps18a: mitochondrial ribosomal protein S18a). Constant expression of Mrps18a was confirmed against other common reference genes. The fold change of mRNA levels was calculated with the relative standard curve method. Measurements were performed in technical triplicates and six biological replicates. Melting curves and gel electrophoresis of the PCR products were routinely performed to determine the specifity of the PCR reaction.