Cfx96 touch deep well

MCF7 and A459 cells were co-incubated with NK cells activated with MU1 (5 mg/ml) with or without IL-2 for 3 days. After treatment, NK cells total RNA was extracted using Thermo Fisher Scientific MagJET RNA Kit, according to the manufacturer’s protocol, and genomic DNA was eliminated using a DNase (QIAGEN). RNA quality was confirmed by the A260/A280 ratio using a NanoDrop (Thermal Scientific) spectrophotometer. Aliquots of 10 ng RNA from each sample were used for subsequent cDNA synthesis. The total cDNA was synthesized using Thermo Fisher Scientific cDNA Synthesis Kit based on the manufacturer’s protocol and PCR machine (biorad). cDNA samples were used to evaluate the expression of INFγ, NKG2D and KIR2DI genes using Maxima SYBR Green/ROX qPCR Master Mix using forward and reverse primer (Supplementary Table S2), SYBR green PCR master mix, according to the manufactured protocol and qPCR machine (Real-Time PCR Detection System Bio-Rad, CFX96 Touch Deep Well). The thermal cycling protocol of RT was as follows: 50°C for 2 min, 95°C for 15 min 40 cycles of 15 s at 94°C, 30 s at 50°C and 30 s at 72°C. RT-PCR were analyzed, and gene expression figures were generated by CFX96 Touch Deep Well, Real-Time PCR Detection System Bio-Rad) software.

Extraction and purification of total RNA were performed using RNAiso Plus (Takara, Japan). Then cDNA was synthesized with random nonamer and oligo dT(18) using PrimeScript II reverse transcriptase (Takara). qPCR was conducted using GoTaq qPCR mix and run on a CFX96 Touch Deep Well (Bio-Rad, Hercules, CA, USA). Relative quantification was done by standard curve method, and the expression levels of target genes were normalized against that of the reference gene, ornithine decarboxylase antizyme (OAZ1).

To verify the accuracy and validity of transcriptome sequencing data, reverse transcription cDNA products were amplified by polymerase chain reaction (PCR). Primers targeted AQP1 (Cluster-1445.13691), AQP2 (Cluster-1445.18746), CREB3 (Cluster-1445.10450), ADCY3 (Cluster-1445.26438), FXYD2 (Cluster-1445.18046), HIF1A (Cluster-1445.20583), and NFATc1 (Cluster-1445.8040). (Supplementary Table S1). Polymerase chain reactions consisted of 10 μM of each primer, dNTP Mixture, and TB Green^® Premix Ex TaqTM Ⅱ (Takara Bio, Beijing, China), in a total reaction volume of 20 μl. Quantitative RT-PCR was performed on a CFX96 Touch Deep Well (Bio-Rad, Delaware, American). Conditions were 95°C for 3 min, 45 cycles of 95°C for 5 s, 60°C for 30 s, and 72°C for 10 s. Analysis of relative gene expression was performed using the 2^−ΔΔCt method (Schmittgen and Livak, 2008 (link)). Values were normalized to GAPDH values and expressed as gene/GAPDH ratio.