Iq sybr green supermix

The qRT-PCR were carried out in 96-wells plates with the iCycler iQ real time PCR detection system (Bio-Rad) using iQ SYBR Green Supermix (Bio-Rad) in a reaction volume of 20 μL containing 10 μL 2X iQ SYBR Green Supermix, 5 μL qRT-PCR template diluted appropriately and 1.5 μL of primer (5 μM). The cycling conditions were as recommended by the manufacturer: 3 min at 95°C preceding 40 cycles (10 s at 95°C followed by 30 s at 60°C). At the end of the run, a melting curve was generated by heating the amplicon from 60 to 95°C in order to confirm the specificity of the amplification for each primer pair. To check the absence of contamination by genomic DNA, PCR reactions on RNA templates were performed for each primer pair. All qRT-PCR were run in technical duplicates and biological triplicates. Standard curves were generated to calculate the PCR efficiency using a fivefold dilution series of a pooled sample containing an equal fraction of all cDNAs of the experiment. Efficiency (E) of each primer pair was obtained from the slope of the calibration curve generated according to the equation:
Primers associated with amplification efficiency below 90% and above 110% were not considered.

qPCR was performed using iQ SYBR Green Supermix (Bio-Rad Laboratories, Hercules, CA, USA), in a total volume of 15 µL, which included 0.2 µg of cDNA, 0.4 µM of each primer, 7.5 µL of iQ SYBR Green Supermix containing dATP, dCTP, dGTP, and dTTP at concentrations of 400 µM each, and 50 units/mL of iTag DNA polymerase. The PCR protocol included 10 min at 95 °C followed by 40 cycles of 10 s at 95 °C (denaturation), annealing for 10 s at a temperature suitable for each gene, and primer extension for 20 s at 72 °C, followed by 10 min at 72 °C. To distinguish specific products from nonspecific products and primer dimers, melting curve analyses were performed. To evaluate the specific mRNA expression in the samples, a standard curve was generated for each run, and three points of the human control cDNA were analyzed (Clontech Laboratories, Inc., Mountain View, CA, USA). The concentration of each sample was calculated using a standard curve.

For quantitative real-time PCR, total RNA was treated with DNase I (TaKaRa, Japan) followed by phenol/chloroform extraction to remove DNA contamination. Approximately 1 μg of purified RNAs was used for first-strand complementary DNA synthesis using PrimeScript Reverse Transcriptase (TaKaRa, Japan) with oligo(dT) primers. Real-time PCR was performed using iQSYBR Green Supermix (Bio-Rad) with primer pairs listed in Additional file 1: Table S1. Relative transcript levels were determined for each sample by normalizing them to ACTIN according to the ∆∆Ct method [46 (link)].
For calculating the relative integrity (RI) value of GUUS and TT4 gene, DNA samples were prepared using CTAB methods. Approximately 50 ng DNA was used for PCR amplification. Real-time PCR was performed using iQSYBR Green Supermix (Bio-Rad, USA) with primer pairs listed in Additional file 1: Table S1. The RI value of GUUS gene were determined by normalizing the Ct Value of U fragment to S fragment according to the ∆∆Ct method and the RI value of TT4 gene were calculated similarly by normalizing the Ct Value of sgRNA-targeted regions to non-targeted regions using primer pairs listed in Additional file 1: Table S1.

Total RNA was extracted from punched samples of the mPFC using an RNeasy kit (Qiagen) followed by cDNA synthesis using a Quantitect Reverse Transcription kit (Qiagen). Real-time polymerase chain reaction (PCR) was performed on three independent sets of templates using iQ SYBR Green Supermix (Bio-Rad). The amplification mixture consisted of 1 μM primers, 10 μl of iQ SYBR Green Supermix (Bio-Rad), and 100 ng of template DNA in a total volume of 20 μl. Cycling parameters were 95 °C for 15 s, 57 °C for 1 min and 72 °C for 40 cycles using a CFX96 real-time PCR detection system (Bio-Rad). For each assay, PCR was performed after melting curve analysis. To reduce variability, we ran each sample in duplicate and included control qPCR reactions without the template in each run.
For microarray analyses, we normalized all microarray data using RMA method provided by the affy package [56 (link)] and analyzed differentially expressed genes using the limma package [57 (link)]. For gene-probe mapping information and GO term annotation, we used the EnsEMBL database (version 90).

Total RNA was extracted with TRIzol™ Reagent (Thermo Fisher Scientific, Carlsbad, CA, USA). RNA yield and purity were determined at 260/280 nm using a Jenway 7315 spectrophotometer (Bibby Scientific, Stone, Staffordshire, UK). An equivalent amount of RNA (2 μg per sample) was used to synthesize cDNA using the MG cDNA synthesis kit (MGmed, Geumcheom-gu, Seoul, Korea). Reverse transcription was performed using random octamer at 65 °C for 5 min and MMLV reverse transcriptase at 42 °C for 30 min. The VEGF-specific primers and β-actin-specific primers were used as reference genes in the experiment, and the results are shown in Table 1. Real-time PCR was performed using iQ™SYBR^®Green Supermix (Bio-rad, Hercules, CA, USA) in accordance with the thermal cycling protocol of iQ™SYBR^®Green Supermix. The thermal cycling protocol conditions included the initial polymerase activation and DNA denaturation step at 95 °C for 3 min followed by the amplification step of 40 cycles consisting of denaturation at 95 °C for 10 s and annealing and extension at 55 °C for 30 s. Melt curve analysis was performed at the end of the amplification step to assess the specificity of the products formed. The relative expression level of VEGF mRNA was normalized to β-actin mRNA and calculated using the 2^−ΔΔCT method. The results were presented as the relative expression compared to control at the same time.