Sensifast probe hi rox kit

RNA was isolated from cell lines or lungs using an E.Z.N.A. Total RNA kit (Omega Bio-Tek, Norcross, GA) and converted to cDNA by high capacity cDNA reverse transcription kit (Life Technologies). Relative expression levels of c-fos, IFNα1, IFNβ1, IFNλ3, Ptges2, Reg3g, and TIMP1 were measured using probes from Applied Biosystems (Grand Island, NY) and a SensiFAST Probe Hi-ROX kit (Bioline, Taunton, MA). Samples were run on a StepOnePlus qPCR machine (Applied Biosystems), and results computed relative to WT uninfected and actin control using the ΔΔC_T method.

Total RNA was isolated from HT-22 cells and primary cultured hippocampal neurons using a RNA purification kit (GeneAll) according to the manufacturer’s instruction. The cDNA was synthesized from 1 μg of total RNA and reverse transcription was performed with SuperScript® VILO™ cDNA Synthesis Kit (Invitrogen). The qRT-PCR was done with a SensiFAST™ Probe Hi-ROX Kit (Bioline) using the TaqMan probe Mm.PT.58.8488376 (IDT). GAPDH was used as an endogenous control. The 2^-ΔΔCt method was used to calculate fold changes in gene expression [[63 (link)]].

Total RNA was isolated with NucleoSpin RNA/Protein kit (Macherey-Nagel) and the quantity of RNA was determined by NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies). The total amount of RNA was 1,000–4,000 ng/sample, which was isolated from 20,000 to 40,000 cells (there was no significant difference between samples from patients and controls). Complementary deoxyribonucleic acid (cDNA) was synthesized from total amount of RNA with a SensiFAST cDNA Synthesis Kit (Bioline) in accordance with the manufacturer’s instructions. The real-time PCRs were carried out in PCR Master Mix containing SensiFAST™ Probe Hi-ROX Kit (Bioline) using TaqMan assays (Thermo Fisher Scientific) for hypoxanthine phosphoribosyltransferase 1 (HPRT-1) (Hs02800695_m1) or RORC (Hs01076122_m1) or TBX21 (Hs00203436_m1) and 25 ng cDNA per gene/well in 8 µL final volume. Specific transcript levels were referred to those of HPRT-1; and the ΔΔC_t calculation method was used to determine the appropriate gene expressions (38 (link)).

Total RNA was defrosted, treated with DNase (1 μg) and either converted into first-strand cDNA using random hexamers and MuLV reverse transcriptase (Life Technologies) for 1 h at 42 °C and cDNA was subsequently heated at 80 °C for 5 min prior to qRT-PCR assay or used in a one-step qRT-PCR (SensiFAST™ Probe Hi-ROX Kit, Bioline) combining the reverse transcription reaction with the qPCR reaction. Human or mouse ENaC, β-actin or GAPDH were quantified by Taqman primers and probes (Hs01013028_m1, Hs99999903_m1 and Hs03929097_g1, or Mm01182998_g1, Mm00607939_s1 and Mm99999915_g1, all from Life Technologies). The qRT-PCR assays were performed in an ABI PRISM® 7000 with the following default PCR parameters set at 50 °C for 2 min, 95 °C for 10 min and then 40 cycles at: 95 °C for 15 s and 60 °C for 1 min. The default parameters for one-step qRT-PCR were: 45 °C for 20 min, 95 °C for 2 min and then 40 cycles at: 95 °C for 15 s and 60 °C for 1 min. Only for A549 cells PrimePCR™ Probe Assays (Bio-Rad Laboratories Ltd, Hemel Hempstead, UK) were used: Human SCNN1A (qHsaCEP0024081 FAM) and Human GAPDH (glyceraldehyde-3-phosphate dehydrogenase, qHsaCEP0041396 Hex). GAPDH mRNA, in comparison with β actin, seemed to be a more suitable reference gene from in vitro transfections because showed minimal changes in expression levels between the individual samples and experimental conditions.

Total RNA were extracted using the Quick-RNA™ Microprep Kit (Zymo Research) and quantified by NanoDrop 2000 (Thermo Fischer). For RT-qPCR analyses, 2 µg of RNA were reverse-transcribed into cDNA using the cDNA Archive kit (Applied Biosystems). Quantitative gene expression was performed using SensiFAST Probe Hi-ROX kit (Bioline, London) on a QuantStudio 5 (Thermo Fischer). Results were normalized to 18S expression and analyzed using the ∆∆Ct method.

Total RNA was extracted using R&A-BLUE Total RNA Extraction Kit (Intron Biotechnology, Korea). cDNA was synthesized using the cDNA Synthesis Kit (Takara Biotechnology, China). Quantitative real-time PCR (qRT-PCR) was performed using SensiFAST Probe Hi-ROX Kit (Bioline, USA) on the LightCycler 96 instrument (Roche, Germany). Relative mRNA expression was calculated using the delta-delta Ct method and normalized to GAPDH. The sequences of primers are listed in Table 1.Table 1

PCR primer sequences

Gene	Primer	Sequence (5′–3′)
DHODH	Forward	CATAATTGGGGTTGGTGGTG
	Reverse	CTTGGGAAGGTTCCAGATCA
UMPS	Forward	GGCTCAGGAGTTGTGAAAGG
	Reverse	CCTGCTTCCAACTGAACTCC
SLC28A1	Forward	AGGTCCTGCCCATCATTGTC
	Reverse	CAAGTAGGGCCGGATCAGTA
SLC28A2	Forward	AATGGGTGTTTGCAGGAGTC
	Reverse	GAAGACCTAGGCCCGAAAAC
SLC28A3	Forward	GACTCACATCCATGGCTCCT
	Reverse	TTCCAGGGAAAGTGGAGTTG
SLC29A1	Forward	CTGCTCCCGTGGAATTTTT
	Reverse	GATGCAGGAAGGAGTTGAGG
SLC29A2	Forward	CCTCCTTCCCTGGAACTTCT
	Reverse	GTTGAGGAGGGTGAAGAGCA
SLC29A3	Forward	GCCAACTTCCTGCTTGTCA
	Reverse	GTGCCTGGGAGTTCCTCATA

MGMT promoter methylation was analyzed with the MethyLight method [23 (link)]. Briefly, gDNA was subject to bisulfide conversion using the Epitect Bisulfite Kit (Qiagen, Hilden, Germany) according to the manufacturer’s recommendations. Quantitative PCR was performed with the SensiFast Probe HiRox Kit (Bioline) and a primer/probe combination specific for the methylated MGMT promoter sequence (Additional file 1). Fully methylated SSSI treated DNA served as calibrator and the collagenase gene 2A1 (COL2A1) served as endogenous control. The percentage of methylated reference (PMR) value was calculated by dividing the MGMT/COL2A1 ratio of the sample by the MGMT/COL2A1 ratio of the SSSI-treated DNA multiplied by 100. Samples with a PMR value >4 were considered as methylated.