Rotor gene q software

Cells were lysed and total RNA was extracted using TRIzol (Invitrogen, USA) according to the manufacturer’s instructions. The RNA quality and concentration were estimated using denatured gel electrophoresis and spectrophotometry respectively. About 500 ng of the total RNA was reverse transcribed into cDNA using a reverse transcription kit (Fermentas, USA) with random hexamers for target genes. cDNA synthesis of miRNAs was undertaken using the Reverse Transcription System Kit (Promega, USA) with miR-specific stem-loop primers (Table 2). Quantitative real-time polymerase chain reaction (PCR) was performed in triplicate using a 40 cycle PCR in Rotor-gene Q real-time analyzer (Corbett, Sydney, Australia). Each real-time PCR reaction contained 5 μl of 2×SYBR Premix Dimer EraserTM (TaKaRa, Japan), 3 pmol of forward and reverse primers respectively, 1 μl template of cDNA and dH₂O up to the final volume of 10 μl, followed by a melting curve analysis to confirm PCR specificity. The average threshold cycle was used for data analysis by Rotor-gene Q software (Corbett, Sydney, Australia). Gene expression levels were normalized against the expression of β-actin and Snord 47(U47) as internal controls for miRNA expression. The 2 ^−ΔΔCtmethod was employed to estimate the relative expression level of each gene. All reactions were run in triplicate.

Herein, real-time PCR seems to be very applicable to detect the changes about the gene expression of the seeded cell on the scaffolds. The assay was performed at 3rd and 7th days of cell culture. For this aim, the total RNA of the cells were extracted by TRIzol reagent (Sigma, cat. no T9424) and their associated cDNA was synthesized accordance with an optimum protocol. For the cDNA production, M-MuLV reverse transcriptase (RT) and Random Hexamer were bought from Fermentas (cat. no 28025013 and N8080127, respectively). For the real-time PCR reactions (Rotor-gene Q software, Corbett), 0.5 µL of cDNA was used for each test sample and the parameters of temperature and time, were set as 94 °C for 3 minutes for the annealing temperature, the conditions of 35 cycles (94 °C for 30 seconds, 62 °C for 45 seconds, 72 °C for 45 seconds) and the extension time of 7-10 minutes at 72 °C. The relative gene expression values were obtained by the comparative ∆∆Ct method.³⁴ The employed real-time PCR master mix was afforded from Fermentas (cat. no 4309155). Moreover, the primer sequences were collected in Table 1. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as the housekeeping control and the other 2 genes of VEGF-A and TGF-β1 were the experimental genes.

Total RNA was extracted from the cells using TRIzol (Invitrogen, USA) according to the manufacturer’s instruction. The quality and concentration of total RNA were subsequently estimated using denatured gel electrophoresis and spectrophotometer, respectively. According to previous study miR could be amplified with stem-loop realtime PCR using specific stem-loop primers (30 (link)). Expression level of the corresponding predicted mRNA targets (Insr and Vamp2) to the selected miR was also validated by real-time PCR using gene specific primers. The threshold cycle average was used for data analysis by Rotor-gene Q software (Corbett, Australia). Genes and related specific primers are presented in the Table 1. Insr and Vamp2 expressions were normalized against the expression of β-actin. Snord-6 (U6) was also selected as internal control for miR expression. All PCRs were performed in triplicates and run in at least 3 independent experiments. The 2^−ΔΔCtalgorithm was employed to evaluate the relative expression level of each gene.

Specimens were aliquoted and frozen at −80°C on the day of collection until processing at the Sunnybrook Health Sciences research microbiology laboratory (Toronto, Canada). RNA extractions were performed using the QIAamp Viral RNA Mini Kit (Qiagen, Venlo, The Netherlands). Reverse transcriptase polymerase chain reaction (RT-PCR) using the Luna Universal Probe One-Step RT-qPCR Kit (New England BioLabs, Ipswich, MA) was performed to detect the 5’ untranslated region and envelope (E) gene of SARS-CoV-2, with RNaseP as internal control. Cycle thresholds (Cts) were determined using Rotor-Gene Q software (Qiagen, Venlo, The Netherlands); results where all targets had Cts <40 were reported as positive.

A SYBR green real-time RT-PCR assay specific for the Horsens strain was designed. The primers were designed to target ORF3, spanning the recombination breakpoint resulting in a 251 bp amplicon. The real-time RT-PCR was run on a Rotor-Gene Q machine (QIAGEN) in a total volume of 25 µL using the Qiagen OneStep RT-PCR kit with 2 µL extracted RNA, 1.5 µM of each primer and 2× SYBR green (SYBR Green I Nucleic Acid Gel Stain 10,000×, CAMBREX) final concentrations. The amplification profile was: 50 °C, 30 min; 95 °C, 15 min; 45 cycles (94 °C, 15 s; 60 °C, 15 s; 72 °C, 60 s); 72 °C, 5 min; 50 °C, 1 min, immediately followed by a melting point analysis, ramping from 50 to 99 °C, rising 1 °C per step, holding 5 s at each temperature increase. The fluorescence signal was measured at 72 °C during each PCR cycle and at each temperature increase during melting analysis. The fluorescent signals were analyzed with Rotor-Gene Q software v. 2.3.1 (QIAGEN), setting the NTC threshold at 10% and cycle threshold at 0.01.

Toal RNA from the treated cells were isolated using Trizol reagent (Invitrogen), and synthesized to cDNA using MuLV Reverse Transcriptase. Real-time PCR were performed on a Rotor-Gene Q real-time PCR cycler (Qiagen, Shanghai, China) using SYBR-green PCR master mix kits as described previously [17 (link), 19 (link)]. The primers were synthesized by Sangon Biotech (Shanghai, China) and the oligonucleotide sequences were as follows: CHOP: 5’-CCACCACACCTGAAAGCAGAA-3’(forward primer), 5’-GGTGCCCCCAATTTCATCT-3’(reverse primer); GRP78: 5’-ACATGGACCTG TTCCGCTCTA-3’ (forward primer), 5’-TGGCTCCTTGCCATTGAAGA-3’ (reverse primer); β-actin: ’-CGGGGACCTGACTGACTACC-3’ (forward primer), 5’-AGGA AGGCTGGAAGAGTGC-3’ (reverse primer). The data were analyzed using the Rotor- Gene Q software (version 1.7, Qiagen), and then relative mRNA levels were calculated by the 2^–△△Ct method.