Mj mini opticon real time pcr system

Total RNA contents were extracted from heart tissue samples in 1 ml QIAzol (79306, QIAGEN Inc., Valencia, CA, USA) with chloroform. The RNA pellets were rinsed with 70% ethanol, dried, and suspended in diethylpyrocarbonate (DEPC, 129112, QIAGEN Inc.). RNA amount and purity were assessed using a spectrophotometer at 260 nm. The ratio of the 260/280 optical density of all RNA tested was 1.7–1.9. RNA in samples was transcripted to the corresponding cDNA with RevertAid Premium reverse transcriptase (EP0733, Thermo Fisher Scientific, Deutschland, Germany). EPO, VEGF, and inducible nitric oxide synthase (iNOS) gene expressions' concentration were examined with RT-PCR using a Bio-Rad MJ Mini Opticon Real-Time PCR System. The primer sequences for EPO, VEGF, iNOS, and GAPDH (housekeeping) genes are listed in Table 1. Data are presented relative to control values using three separate experiments.

Real-time quantitative reverse transcription PCR (qRT-PCR) analysis was performed with MJ MiniOpticon Real-time PCR system (Bio-Rad, Hercules, CA, United States) using SsoFast™ EvaGreen supermix (Bio-Rad) in 15 μl reaction volume. The PCR conditions were 95°C for 30 s (initial denaturation) followed by 40 cycles at 95°C for 5 s, and 60°C for 10 s. The program was further followed by a melting curve analysis ranging from 65°C to 95°C with an increment of 0.5°C every cycle. All analyses were performed with three biological replicates and two technical replicates. The results were analyzed using CFX connect software (Bio-Rad) using 2^(−ΔΔCt) method. The relative expression levels were normalized with housekeeping genes, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) or Actin. Primer sequences of all the genes used in this study are listed in Supplementary Table 1.

Total RNA from the cells was extracted using TRIzol reagent (Invitrogen, United States) according to the manufacturer’s protocol. A UV spectrophotometer was used to measure the purity and concentration of the RNA, and agarose gel electrophoresis was used to verify the RNA integrity. The cDNA templates were synthesized using the PrimeScript^TM RT reagent kit with gDNA Eraser (TaKaRa, Japan) according to the manufacturer’s instructions. The qPCR analysis was performed using IQ SYBR Green Supermix (Bio-Rad, United States) and the Bio-Rad MJ Mini Opticon Real-Time PCR System, along with Bio-Rad CFX Manager analysis software. The resulting amplification and melting curves were analyzed to identify the specific PCR products. The relative gene expression values were calculated using the comparative 2^-ΔΔCt method, and GAPDH was used as control. The PCR primers for BDNF were as follows: forward primer: 5′-GGGACCCGTGAGTTTGTGT-3′, and reverse primer: 5′-TTGCTTCTTTCATGGGGGCA-3′. The PCR primers for GAPDH were as follows: forward primer: 5′-GACAGTCAGCCGCATCTTCT-3′, and reverse primer: 5′-GCGCCCAATACGACCAAATC-3′.

Total RNA was extracted from freshly isolated colonic tissues as well as HT-29-Cl.16E cells treated with recombinant VIP (0.6μM, 1μM and 3μM) by using Qiagen RNeasy plus mini kit. RNA was reverse-transcribed using Superscript II reverse transcriptase (Invitrogen) and qPCR and quantification was carried out as previously described [17 (link)]. Quantitative PCR was carried out on a Bio-Rad MJ Mini-Opticon Real-Time PCR System (Bio-Rad), using IQ SYBR Green Supermix (Bio-Rad). Individual primer sequences are listed in Supplementary Information (S1 Table). After completion of the cycling process, samples were subjected to a temperature ramp (from 53 to 95°C) with continuous fluorescence monitoring for melting curve analysis. For each PCR product, a single narrow peak was obtained by melting curve analysis at the specific melting temperature, indicating specific amplifications. Primer pair efficiency was tested according to manufacturer's instructions. Quantification was carried out with Gene Ex Macro OM 3.0 software (Bio-Rad) where PCR efficiencies for each of the primer sets were incorporated into the final calculation. The ΔΔCt method was used to calculate the relative amount of specific RNA present in a sample, from which the fold induction of transcription of the gene was estimated by comparison to values relative to the control samples. Data are expressed as means ± SD.

TRIzol reagent (Invitrogen, CA) was used to extract total RNA from the cells according to the manufacturer’s instructions. First-strand cDNA was acquired by reverse transcription using a cDNA synthesis kit (Fermentas, CA) according to the manufacturer’s instructions. The cDNA was stored at −20 °C until use. qPCR was performed using IQ SYBR Green Supermix (Bio-Rad, USA) and the Bio-Rad MJ Mini Opticon Real-Time PCR System along with the matching analysis software Bio-Rad CFX Manager. The resulting amplification and melt curves were analysed to ensure the identity of the specific PCR product. Relative gene expression levels were calculated by normalization to GAPDH. The sequence of each designed primer is as follows:
Mouse GAPDH primer
Sense: 5′-ACAACTTTGGCATTGTGGAA-3′,
Antisense: 5′-GATGCAGGGATGATGTTCTG-3′;
Mouse DNMT1 primer
Sense: 5′-CTGCTGTGGAGAAACTGGAA-3′,
Antisense: 5′-TGATTTCCGCCTCAATGATA-3′;
Mouse DNMT3β primer
Sense: 5′-CTCAAACCCAACAAGAAGCA-3′,
Antisense: 5′-AGCAGCAGAGTCATTGGTTG-3′;
Trp53 primer
Sense: 5′-CTCAGACTGACTGCCTCTGC-3′,
Antisense: 5′-GGCTGAGCCCTAGCTACAAG-3′.