Lightcycler 96 application software

Total RNA was isolated from the indicated strains grown to log-phase at 25ºC in synthetic complete media or further incubated at 37ºC for 2 h, as described previously [25 (link)]. cDNA synthesis was performed on 2 μg total RNA using random hexamers (Invitrogen) and Rever Tra Ace (Toyobo) according to the manufacturer’s protocol. RT-qPCR was performed on 1:5 diluted cDNA using a Light Cycler 96 system (Roche) with 1x THUNDERBIRD qPCR Mix (Toyobo) in a total reaction volume of 10 μL containing 0.3 μM of the appropriate primer pair. The results were analyzed using Light Cycler 96 Application Software Version 1.1 (Roche). After the PCR reaction, amplification specificity was confirmed by melt-curve analysis. The relative amounts of RNA in each sample were calculated for three independent biological replicates using the ΔCq method. The primer pairs used were as follows: PGK1, TK13695-TK13696; VTC1, TK13936-TK13937; VTC1^#, TK14458-TK13937; SCR1, TK13934-TK13935.

Total cellular RNA was extracted with TRIzol reagent (Invitrogen) and the Total RNA Extraction Kit (TIANGEN Biotech), the concentration of which was measured with a spectrophotometer. Quantitative real-time PCR (qRT-PCR) was performed with PrimeScript RT Master Mix (TaKaRa) according to the manufacturer’s protocol. Each cDNA sample was denatured at 95 °C for 5 min and amplified for 35 cycles of conditions including 15 s at 98 °C, 30 s at 58 °C, and 30 s at 72 °C with a LightCycler 96 (Roche). The mRNA expression level of each target gene was normalized to the respective GAPDH and analyzed using LightCycler® 96 Application Software (Roche). The qRT-PCR primers are listed in Supplementary Table 3. Notably, five pairs of qRT-PCR primers for the newly identified lncRNAs by RNA-seq were designed and applied. The suitable primers were screened with stable results from three independent experiments and are listed in Supplementary Table 3. Northern blotting was performed using a NorthernMax Kit (Ambion) with biotin-labeled probes, the sequences of which are shown in Supplementary Table 3. The viral load of HTNV was quantified based on the qRT-PCR and absolute quantitative PCR.

The methylation level of MGMT promoter was determined by MS-HRM on Light Cycler®96 (Roche Diagnostics GmbH, Germany) in T98G cell line. First, 500 ng of genomic DNA was bisulfite converted using EZ DNA Methylation Kit (Zymo Research, USA). As positive/methylated and negative/unmethylated controls, we used CpG Methylated HeLa Genomic DNA (New England Biolabs, USA) and CpGenome Universal Unmethylated DNA Set (Merck, Germany), respectively. Standardized solutions with a given DNA methylation percentage (100, 75, 50, 25, 15, 10, 0% of methylated controls in a background of unmethylated DNA) were prepared. MS-HRM was performed using 5 X Hot FIREPol EvaGreen HRM Mix (Solis BioDyne Co., Estonia) under default cycling conditions and with primers previously published by Wojdacz & Dobrovic [26 (link)]. After amplification, the HRM profiles were analyzed using Light Cycler®96 Application Software (Roche Diagnostics GmbH, Germany). Samples were run in duplicate in each experiment, and the experiment was repeated twice.