Miniopticon real time pcr system

To validate the RNA-Seq analysis, RT-qPCR was performed on a set of eight randomly selected genes. DNase I treated RNA samples were reverse transcribed using First Strand cDNA Synthesis Kit (Thermo Scientific). Primers for RT-qPCR were designed using the Primer3Plus online software (www.bioinformatics.nl/primers3plus) and their sequences are available in S1 Table. The RT-qPCR reactions were performed with the MiniOpticon Real-Time PCR System, Bio-Rad (Hercules, CA, USA) and the SYBR Green 2x Master Mix (Ampliqon) were used to detect transcript abundance. The reaction was performed using 1.5μl of first strand cDNA, 3μl of each primer and 7.5 μl SYBR Green Master Mix in a final volume of 15 μl. Negative control was also considered for each run. Cycling programs were incubation at 95 °C for 5 min, then 40 cycles of denaturation at 92 °C for 45 s, annealing at 60 °C for 45 s and extension at 72 °C for 45 s. The specificity of all products was verified via melting curve analysis by increasing the temperature from 60 °C to 95 °C and read every 0.5 °C. Three replicates were considered for each gene. Normalization of reads was done concerning the Actin 2 as the reference gene. The relative quantitative method (2^-ΔΔCt) was used to estimate quantitative gene expression.

Total RNA was extracted from HUVECs that were treated with vehicle or brassinin using a RNeasy Mini kit (Qiagen, Hilden, Germany). Then, equal amounts of total RNA (1 μg) were reversely transcribed using a QuantiNova Reverse Transcription Kit (Qiagen). Quantitative real-time PCR was conducted using a QuantiNova SYBR green PCR kit (Qiagen) on a BioRad MiniOpticon Real-Time PCR System. The messenger RNA (mRNA) levels of genes were calculated using the 2^−ΔΔCt method with GAPDH as an endogenous control and expressed as a percentage of vehicle-treated control group. The specific primer sequences are listed as follows: 5′-TTAGCCAGCTTAGTTCTCTGTGG-3′ (forward) and 5′-AGCATCAGATACAAGAGGTAGGG-3′ (reverse) for human Tie2; 5′-GGCTACAAGGTCCGTTATGCC-3′ (forward) and 5′-GATGCTGCCGTACTCATTCTC-3′ (reverse) for human FGFR1; 5′-ATGGGTGTGAACCATGAGAAGTA-3′ (forward) and 5′-GGCAGTGATGGCATGGAC-3′ (reverse) for human GAPDH.

The Odvill mRNA of gills was detected by the SYBR Green PCR Master Mix (Bio-Rad Laboratories, Hercules, CA, USA) and quantified with the Mini Opticon real-time PCR system (Bio-Rad). The Odvill mRNA values were normalized using the expression of the β-actin mRNA from the same DNA samples. One identical cDNA sample from the 50% SW-acclimated fish was used as the internal control among different groups. For each unknown sample, the comparative Ct method with the formula 2^-[(Ct_Odvill,n-Ct_β-actin,n)-(Ct_Odvill,c-Ct_β-actin,c)] was used to obtain the corresponding Odvill and β-actin values, where Ct corresponded to the threshold cycle number.

DPSCs in hypoxic condition (Table 1) were analyzed by RT-qPCR analysis. Total cellular RNA was extracted from 100,000 DPSCs using TRIzol^® Reagent (Thermo Fisher Scientific, Rockford, IL, USA), and its quality and quantity were evaluated on a NanoDrop spectrophotometer (Thermo Fisher Scientific, Rockford, IL, USA). For the qPCR assay, cDNA was synthesized from 500 ng of total RNA with SuperScript (Euroclone, Milan, Italy). Next, the qPCR was carried out using SYBR green master mix (Luna), according to the manufacturer’s protocols. The amplification reaction was performed on a MiniOpticon Real-Time PCR System (Bio-Rad, Milan, Italy) using the following program: the RT reaction was set at an initial denaturation step at 95 °C for 1 min, followed by 95 °C for 15 s. The reaction mixture was heated according to the Tm shown in Table 3 for 30 s, followed by amplification that consisted of 40 cycles of denaturation at 95 °C for 15 s, and annealing and extension at 60 °C for 30 s, according to the manufacturer’s protocol. The relative expressions of the neuronal marker genes investigated were calculated using the relative quantification 2^{(−∆∆C(T))} 2^−∆∆Cq method [58 (link)], with GAPDH as a reference gene commonly used for this purpose. The primer sequences used in this RT-qPCR analysis are listed in Table 3.

RNA extraction and reverse transcription were carried out as previously described (22 (link)). RNA quality and quantity were assessed by spectrophotometry (NanoDrop ND-1000; Thermo Fisher Scientific, Waltham, MA, United States) and its integrity was determined with an Agilent 2,100 Bioanalyzer (Agilent Technologies Inc., Waldbronn, Germany). In all cases, the RNA integrity was ≥9 and the A260/280 ratio was between 1.96 and 2.02. Total RNA was reverse-transcribed using an iScript™ cDNA Synthesis Kit (Bio-Rad, Hercules, CA, United States). For real-time PCR determinations, we used a cDNA template in a 20-μL reaction solution containing 0.2 μmol/L of each primer and SsoAdvanced™ Universal SYBR^® Green Supermix (Bio-Rad). The primers used are listed in Table 2. Real-time PCR was performed on a MiniOpticon Real-Time PCR System (Bio-Rad). Each PCR run included duplicates of reverse transcript cDNA for each sample and negative controls (reverse transcription-free samples, RNA-free sample). Quantification of the target gene transcripts was conducted using glucuronidase beta (Gusβ) gene expression as reference and was performed with the 2^−ΔΔCT method (23 (link)). Product fidelity was confirmed by melting curve analysis.