Sensiscript reverse transcriptase kit

Total RNA was extracted from PBMCs and oral epithelial cells using an RNeasyPlus Mini Kit (Qiagen, Valencia, CA, USA), and reverse transcription was performed with a Sensiscript Reverse Transcriptase Kit (Qiagen). The primers used in the real-time PCR assay are detailed in Supplementary Table 1.
GAPDH and β-actin mRNA levels were used to normalize mRNA content from the PBMCs and oral epithelial cells, respectively. PCR was performed in an Applied Biosystems 7500 system using specific primers and SYBR Green fluorescence detection reagents (Applied Biosystems, Carlsbad, CA, USA). The cycling protocol consisted of 10 min at 95 °C, followed by 40 cycles of 15 s at 95 °C and 60 s at 60 °C. The amplification results were visualized and analyzed using Sequence Detection System (SDS) software (Applied Biosystems). Normalized expression was calculated as described previously by Livak32 (link).

RT-PCR was used to analyze the amount of mRNA expression for both Nrf2 and Keap1. The liver, kidney, and spleen homogenates were treated with the triazole reagent and subjected to the standard protocol for isolating total RNA. A Sensi-Script Reverse Transcriptase Kit (Qiagen Hilden, Germany) was used to convert RNA to cDNA. With the use of TaqMan probes, we were able to conduct quantitative PCR. TaqMan Gene Expression Master Mix was used for qPCR using TaqMan primers for mouse Nrf2 and Keap1. The CFX384 Touch Real-Time PCR Detection System (BioRad, Mississauga, ON, Canada) was used for the quantitative RT-PCR. Using the delta–delta Ct technique, we calculated the abundance of each transcript. All reactions were performed in triplicate and the mean value was used to calculate expression levels after normalization against β-actin.

Total RNA was extracted from PBMCs using an RNeasyPlus Mini Kit (Qiagen, Valencia, CA, USA), and reverse transcription was performed with a Sensiscript Reverse Transcriptase Kit (Qiagen). The primers used in the real-time PCR assay for NKG2C, NKG2D, MICA, HLA-E, and UBLPB-3 are shown in Supplementary Table 2.
GAPDH mRNA levels were used to normalize the mRNA content from PBMCs. PCR was performed on an Applied Biosystems 7500 system using specific primers and SYBR Green (Applied Biosystems, Carlsbad, CA, USA) fluorescence detection reagents. The cycling protocol consisted of 10 min at 95°C, followed by 40 cycles of 15 s at 95°C and 60 s at 60°C. The amplification results were visualized and analyzed using Sequence Detection System (SDS) software (Applied Biosystems). Normalized expression was calculated as previously described by Livak [63 (link)].

Viral DNA/RNA protected from digestion within viral capsids were extracted using QIAamp Viral RNA mini kit (QIAGEN), according to manufacturer’s recommendations. A second DNase/RNase step was performed on the extracted viral RNA for elimination of genomic DNA, using DNase I recombinant, RNase free (10U/μl) (Roche) and Protector RNase inhibitor (40 U/μl) (Roche). After digestion of the DNA, the viral RNA was transcribed with Sensiscript Reverse Transcriptase kit (QIAGEN; Sensiscript RT kit) to generate cDNA, according to manufacturer’s instructions with minor modifications. Briefly, for a more sensitive detection in the subsequent PCR, a mixture of oligo-dt primers (Oligo (dT)15 primer, Promega) and random primers (Random hexamers, TaqMan Reverse Transcription Reagents, Roche, Applied Biosystems) were used and a RNA denaturation step (95° for 3 minutes) was added.

DNA and RNA were isolated from FFPE sections using the AllPrep DNA/RNA FFPE kit (Qiagen). Isolated RNA was treated with DNase I (Qiagen) to eliminate potential DNA contamination prior to downstream analysis. cDNA was synthesized using random primers (Ambion, Life Technologies) and the Sensiscript Reverse Transcriptase kit (Qiagen). For each tumor, signature genes found to be upregulated from normative range kidney standards were identified by the tumor gene panel screen and utilized to monitor cell phenotype over time in 2D and 3D cultures. PCR for selected markers was carried out with appropriate SYBR or Taqman assay protocols as described above. Relative changes in gene expression were analyzed by the comparative ΔΔC_T method normalized to the cells on the day of seeding.

Dulbecco’s Modified Eagle’s Medium (DMEM), fetal bovine serum (FBS), SYBR Green, penicillin, streptomycin, L-glutamine, trypsase, dimethlsulfoxide (DMSO), Triton X-100, phenylmethanesulfonyl fluoride (PMSF), â-mercaptoethanol (â-ME), TNF-α detection kit, IL-6 ELISA kit, and cell cycle assay kit were obtained from Sigma-Aldrich (St. Louis, MO, USA). Mouse anti-HCMV IE and mouse anti-HCMV UL83 were purchased from Abcam, USA. Secondary antibody goat anti-mouse and Platinum® Quantitative PCR SuperMix-UDG were obtained from Invitrogen, USA. RNeasy Mini Kit and Sensiscript Reverse Transcriptase Kit were purchased from Qiagen (Valencia, CA, USA).

Total RNA was isolated at OakLabs GmbH. TaqMan® Reverse Transcription Reagents Kit (Thermo Fisher, USA) was used for cDNA synthesis with more than 50 ng per reaction. In contrast, Sensiscript Reverse Transcriptase Kit (Qiagen, Germany) was used for cDNA synthesis with less than 50 ng per reaction. Primers were designed using Primer Blast (NCBI, USA). Probes (6FAM as reporter dye, BBQ as a quencher) were created using the designing tool IDT DNA (Integrated DNA Technologies, USA). Both, primer and probes were synthesised by TIB MolBiol (Berlin, Germany). Sequences are summarised in Supplementary Table 2. Quantitative PCR (qPCR) was performed in the Stratagene Mx3000PTM (Agilent Technologies Inc., USA) using the Brilliant III Ultra-Fast QPCR Master Mix (Agilent Technologies, USA) according to the manufacturer’s instructions with the following temperature profile: 3 min of initial denaturation at 95 °C and 50 cycles of 20 s at 95 °C and 20 s at 60 °C. All values were generated in duplicate and were corrected concerning efficiency.