Rotor gene q hrm

Peripheral venous blood samples from the subjects analyzed were processed within 2 h from the collection by centrifugation into EDTA-coated tubes at 2,000 g at 4°C for 20 min. Plasma samples were then stored at −80°C until further analyzed.
Total RNA was extracted from 100 μl of plasma-EDTA using the total RNA Purification kit from Norgen Biotek Corporation (Thorold, ON, Canada) according to the manufacturer’s guidelines. Circulating miRNA levels were analyzed as described in Olivieri et al. (2014 (link)) using a modified real-time approach with the TaqMan miRNA reverse transcription kit and the miRNA assay from Applied Biosystems (Foster City, CA, USA). qPCR reactions were performed on a RotorGene Q HRM instrument (Qiagen, Germany). The plasma levels of circulating miRNAs are reported as relative expression (RE) normalized to the mean of spiked-in synthetic non-human cel-miR-39. The relative expression of each miRNA was reported as 2^−ΔCt, with ΔCt being the difference between the Cts of a specific miRNA and those of the cel-miR-39. Cel-miR-39 was used also for the assessment of miRNA recovery from biological samples. Only those samples with a cel-miR-39 recovery higher than 95% were processed for inflamma-miRNA quantification. Each reaction was performed in duplicate.

Total RNA was isolated from cells using the RNeasy Mini kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. The first-strand cDNA synthesis kit ReverTra Ace qPCR RT master mix (TOYOBO, Osaka, Japan) was used for cDNA synthesis, and reverse transcription was performed according to the manufacturer’s instructions. Real-time PCR was performed using the Rotor-Gene Q HRM (Qiagen) and the Thunderbird SYBR qPCR mix kit (TOYOBO) according to the manufacturer’s instructions. The relative gene expression was calculated using the delta-delta Ct method. Ct values were normalized against the elongation factor-1α used as an internal control. The primer sequences are listed in Table 1 [30 (link)].

A qPCR master mix (PerfeCTa Sybr Green Fastmix, QuantaBio, Beverly, CA, USA) was used, containing all necessary components for the qPCR reaction except for primers. The reaction mixture consisted of 6.50 µL PCR master mix, 0.87 µL of each primer (333 nM final concentration), 3.89 µL distilled H₂O and 0.87 µL target DNA. The qPCR (using a Rotor-gene Q HRM from Qiagen, Venlo, The Netherlands) was optimized for standard cycling conditions with an initial 240 s enzyme activation step at 95 °C, followed by 30 cycles of 30 s annealing and extension at 60 °C and 5 s denaturation at 95 °C. Afterwards a HRM was conducted by increasing the temperature from 60 °C to 95 °C (ramp speed = 0.5 °C/s).

qPCR/HRM was recorded with the supplied program of Rotor-gene Q HRM and analyzed with the supplied Rotor-gene Q series Software version 2.3.1 (Qiagen, Venlo, The Netherlands). The data analysis (first derivative dF/dt and the threshold) was automatically calculated by this program.
FO-PCR-MA data acquisition was done with the in-house developed LabView program (National Instruments, Austin, TX, USA) to control two spectrometers and the NiDaq coupled thermocouples. By combining the SPR and thermocouple data, a first order derivative could be calculated for each PCR melting cycle. The resulting melting peak was fitted in Matlab (MathWorks, Natick, MA, USA) using a Gaussian fit to determine the T_m and evaluate the melting peak shape for each PCR cycle. The threshold was calculated as x_NTC + 3 σ_noise, where x_NTC is the average value of the non-template control (n = 40 cycles) and σ_noise equals one standard deviation.
The intraclass correlation coefficient (ICC) [30 ], which assesses the reliability of ratings by comparing the variability of different ratings of the same object, was calculated with the statistical package R (version 2.11.1, R foundation for Statistical Computing, Vienna, Austria).

Total RNA was isolated from cultured ESCs using the RNeasy Minikit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. The first‐strand cDNA synthesis kit ReverTra Ace qPCR RT Master Mix (Toyobo, Osaka, Japan) was used for cDNA synthesis. Reverse transcription was performed according to the manufacturer's instructions. Real‐time PCR (RT‐PCR) was performed using the Rotor‐Gene Q HRM (Qiagen) and the Thunderbird SYBR qPCR Mix kit (Toyobo), according to the manufacturer's instructions. The PCR efficiency (E%) for the amplification of each gene was calculated using the following formula: E% = [−1 + 10(−1/α)] × 100, where α is the slope of the corresponding amplification plot.²³ For relative quantification, data were normalized against elongation factor‐1α (EF‐1α) as an internal control. The validated primer sequences are listed in Table 1.

Total RNA was extracted from the cells using a Maxwell RSC simplyRNA Cells Kit (Promega, Madison, WI, USA). Reverse transcription was carried out in accordance with the manufacturer’s instructions using a first-strand cDNA synthesis kit called ReverTra Ace qPCR RT master mix from TOYOBO in Osaka, Japan. Using a Thunderbird SYBER qPCR mix kit (TOYOBO) and Rotor-Gene QHRM (Qiagen, Hilden, Germany) in accordance with the manufacturer’s guidelines, real-time PCR was carried out. The Ct technique was used to calculate the relative gene expression. The elongation factor (EF)-1, which was utilized as an internal control, was used to standardize Ct readings. The list of primer sets used for RT-PCR is displayed in Table 1.