Itaq universal one step rt qpcr kit

As described in our previous study [23 (link)], a gene expression study was performed using qRT-PCR. A complete list of primers is provided in Table 1. U251 cells were seeded in a 6-well plate at a density of 1 × 10⁵ cells/well and cultured in DMEM + 10% (v/v) FBS until their confluence reached 90%. Once the target confluence was reached, the medium was replaced with fresh 75% (v/v) hMSC-CMs in DMEM + 10% (v/v) FBS and the cells were cultured for another 72 h. U251 cells cultured in 75% (v/v) U251-CMs in DMEM + 10% (v/v) FBS served as controls. At the end of the culture, total RNA was isolated from treated U251 cells using TRIzol™ reagent (Sigma-Aldrich, U.S.A.), according to the manufacturer’s instructions. The isolated RNAs were then used to determine the expression levels of target genes by iTaq Universal One-Step RT-qPCR Kits (Bio-rad, U.S.A.), according to the manufacturer’s instruction. PCR was carried out using an Applied Biosystems 7500 Fast Real-Time PCR system (Applied Biosystem, U.S.A.) with the following setting: initial denaturation at 95°C for 10 min, followed by 40 cycles of denaturation (95°C, 10 s), annealing (60°C, 10 s), and extension (72°C, 40 s). The mRNA level of each target gene was normalized with the mRNA level of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) using a 7500 software version 2.0.5 (Applied Biosystem, U.S.A.).

Total RNA was extracted with an RNeasy Plant Mini Kit (Qiagen). First-strand cDNA was synthesized from 1 μg RNA using a PrimeScript™ RT reagent Kit with gDNA Eraser (Takara). qRT-PCR was performed using a Bio-Rad CFX384 and iTaq Universal One-Step RT-qPCR Kits (Bio-Rad) according to the manufacturer’s instructions. Results were calculated using Bio-Rad CFX manager software. Tubulin was used as internal control. The relative expression of genes was calculated using the ΔCt method. The primer pairs for qRT-PCR were designed by Primer3Plus (http://www.primer3plus.com) (Supplementary Table 4) and compared by BLAST analysis to the NCBI database for confirmation of primer specificity.

An RNA standard was constructed by initially reverse transcribing the CTV RNA. Subsequently, a 349 nucleotide-long sequence from the CTV ORF1 helicase region between nucleotides 3217 and 3565 was amplified and cloned into a pDrive vector (Qiagen, Düsseldorf, Germany). Following transformation into XL10 Gold cells, the plasmid containing the specific insert was isolated and linearized, and the region of interest was transcribed using a T7 High Yield RNA Synthesis Kit (New England Biolabs, Ipswich, MA, USA). Prior to quantification, CTV RNA was extracted using the QIA-amp Viral RNA Kit (Qiagen). The extracted samples were prepared for quantification using the iTaq Universal One-Step RT-qPCR Kit (Bio-Rad, Hercules, CA, USA). The primers used amplify the genomic region between nucleotides 3247 and 3409: (Forward) 5′-ggcaaccatcctctacaaacac-3′ and (Reverse) 5′-gatgtcttgtgggagcctgtag-3′. The thermal cycling conditions were 50 °C for 10 min, 95 °C for 1 min, and 40 cycles of 95 °C for 10 s, 65 °C for 20 s, and 72 °C for 40 s.

The validity of differential expression was verified by using RT-qPCR for direct comparison with RNA Seq. The qPCR reactions (10 uL) were performed in triplicate using iTaq Universal One-Step RT-qPCR kit (BioRad, Hercules, CA, USA) with 0.5 μM of each primer (Supplementary Table 2), and 1 ng of total RNA as template. First cDNA was synthesized by reverse transcription at 50 °C for 10 min followed by reverse transcriptase inactivation at 95 °C for 1 min. The reaction was directly followed by PCR amplification as follows: 40 cycles of denaturation: 30 s at 95 °C; annealing: 30 s at 55 °C; and extension: 30 s at 72 °C. The final PCR step was 30 s at 96 °C followed by 5 s at 60 °C and the PCR reaction was stopped by a constant temperature of 4 ℃. The relative expression of the target genes was calculated using gyrA and rpoA as reference genes and using the efficiency-corrected REST model [37 (link)], CFX Maestro software version 2.2 (BioRad) and qbase + version 3.3 [36 (link)]. The gyrA and rpoA genes were chosen using reference gene selection tool CFX Maestro software version 2.2 (BioRad). For each comparison, four biological replicates and three technical replicates were used for all calculations.