Rotor gene q real time pcr

The total RNA was isolated from cells using Tripure Isolation Reagent (Roche Diagnostics GmbH, Mannheim, Germany) following manufacture’s instruction. Reverse transcription was performed using iScript cDNA Synthesis Kit (#1708891, Bio-Rad, Hercules, CA, USA) for cDNA synthesis and the quantitation of gene expression was achieved from Luna^® Universal quantitative Polymerase Chain Reaction (qPCR) Master Mix (#M3003, New England Biolabs, Ipswich, MA, USA) following manufacture protocol. Quantitative PCR reactions were performed using a QIAGEN Rotor Gene Q Real-Time PCR. The primer sequences (5′–3′; forward, reverse) used in qPCR was showed in Table A1. Amplification was achieved for 40–50 cycle following enzyme activation step in 95 °C for 1 min hold, denaturation in 95 °C for 15 s, and extension step in 60 °C for 30 s. The expression of target genes was normalized to internal control β-actin. The fluorescence signal was detected at the end of each cycle. Melting curve analysis was used to confirm the specificity of the products. The data were calculated using the comparative methods of the −ΔCt (-delta Ct) or 2^−ΔΔCt (standardized mRNA level).

Real-time qPCR for ALU, LINE-1, β-actin, and GAPDH genes using SYBR green chemistry was used to determine the concentration of large (>205 bp) and small (110 bp) fragments of cfDNA in the sera of healthy, CLD, and HCC patients. RT-qPCR was carried out in a 10 µL final reaction volume using the Rotor-Gene Q real-time PCR (Qiagen) System. Each reaction mixture contained 2 µL (∼10 ng) of cfDNA template, 0.2 µL forward and reverse primer (10 µM), 5 µL 2X SYBR green (Agilent), and 2.4 µL nuclease-free water (Qiagen). The reaction condition was 95°C for 5s (seconds), followed by 40 cycles of 95°C for 15 s, annealing (58°C, 52°C, 55°C, and the 50°C) for 20s, and extension at 72°C for 30s. The Ct value of the RT-qPCR test was used to determine the absolute concentration of fragments by interpolating it from the genomic DNA standard curve of known DNA concentrations ranging from 20 ng to 0.02 pg. The concentration of smaller fragments relative to larger fragments was determined by the comparative Ct (2 ^–ΔΔCt) method. Normalization using the mean Ct value of Huh-7 cell genomic DNA was carried out. Assuming a small fragment as target and a large fragment as a calibrator, normalization was carried out using a reference Ct value of Huh-7 cell genomic DNA or healthy control cfDNA.

Total RNA was prepared using Trizol reagent (Invitrogen, Waltham, MA, USA) according to the manufacture’s protocol. Total RNA (1 μg) was used in the reverse transcription (RT) reaction by iScript cDNA Synthesis Kit (Bio-Rad, Hercules, CA, USA). The quantitative real time PCR was performed using KAPA SYBR^® FAST qPCR Master Mix (2X) Kit (KAPA Biosystem, Wilmington, DE, USA) according to the manufacturer’s protocol. Quantitative PCR reactions were performed using a QIAGEN Rotor Gene Q Real-Time PCR. β-actin was used as the endogenous reference gene since it has the highest stability across pig tissues compared to the other reference genes [31 (link)]. The primer sequences (5′–3′; forward, reverse) were showed in Table 1. The amplification steps were set for 3 min at 95 °C, followed by 40 cycles of denaturation at 95 °C for 3 s, and annealing at 60 °C for 20 s. The data were calculated using the standardized mRNA level comparative methods 2^−ΔΔCt. The 2^−ΔΔCt method is a convenient way to analyze the relative changes in gene expression with a high efficiency qPCR assay [32 (link)].

Total RNA was extracted from frozen tomato root tissues using the Spectrum Plant Total RNA Kit (Sigma Aldirch). Then, 1 µg of RNA was treated with 1ug of DNAse RQ1 (Promega, Madison, WI, USA). The cDNA synthesis was performed using ImProm-II Reverse Transcription System (Promega, Madison, WI, USA). Quantitative Real-Time RT-PCR was performed using gene-specific primers (approximately 20 nt-long), amplifying fragments of about 100 bp (Table S1). The specificity of each primer was tested, firstly performing a BLAST directly on the tomato genome mRNA prediction and subsequently carrying out the melting curve. The elongation factor and the ubiquitin primers were obtained by Vigani et al. [14 (link)]. The Real-Time RT PCR was performed using SsoFast EvaGreen Supermix (Bio-Rad, Hercules, California 94547, USA), the Qiagen Rotor Gene Q Real-Time PCR. The amplification efficiency was obtained through LinReg PCR [78 (link)] and the relative expression and the standard error was calculated according to Paffl, Horgan and Dempfle [79 (link)].

The RNA extraction was performed using Spectrum Plant Total RNA Kit (Sigma-Aldrich) after grinding the root tissues in liquid nitrogen. The quality and purity of the samples were obtained spectrophotometrically and by electrophoresis. Then 1 μg of total RNA was subjected to DNAse digestion with 10 U of DNAse RQ1 (Promega) and cDNA was synthetized using ImProm-II Reverse Transcription System (Promega). The amplification primers were designed to be 20 bp long and to obtain an amplicon of 100 bp (Supplementary Table S1). The Elongation Factor 1α and Tubulin housekeeping primers were obtained from Pii et al. (2014) . The Real-Time Reverse Transcriptase PCR (Real-Time RT PCR) was performed using the SsoFast EvaGreen Supermix (Bio-Rad) and the Qiagen Rotor Gene Q real-time PCR. The relative expression and the standard error was calculated according to Pfaffl et al. (2002) (link). The amplification efficiency was calculated using LinRegPCR (Ramakers et al., 2003 (link)).

Two-hundred nanograms of RNA for each sample were retrotranscribed with M-MuLV-RH First Stand cDNA Synthesis Kit (Experteam, Venice, Italy). The expression levels of PAPPA were evaluated with qPCR using FastStart Essential DNA Green Master Mix (Roche, Basilea, Switzerland) on the Rotor-Gene Q real time PCR (Qiagen, Hilden, Germany). Each sample was run in triplicate and normalized against actin (ACTB, Eurofins, Luxemburg, Luxemburg). The quantification of the PAPPA expression levels was determined using the 2-ΔCT method. The specific primers were PAPPA PF: 5′-TGAATCTGAGCAGCACATTG-3′ and PR: 5′-CATCGTCTTCCAAGCACTTC-3′; and ACTB PF: 5′-CACCAACTGGGACGACAT-3′ and PR: 5′-ACAGCCTGGATAGCAACG-3′.