Kapa sybr fast one step rt qpcr kit

The HCT-116 cells were treated with A. elliptica fruit extract (10 µg/mL) or embelin (20 µg/mL) solutions in a serum-free medium for the indicated time. After treatment, RNA extraction was performed by using the RNeasy Mini kit (Qiagen). Real-time quantitative reverse transcription PCR (real-time qRT-PCR) system was performed using the KAPA SYBR FAST One-step RT-qPCR kit (KAPA biosystems, Wilmington, DE, USA) and Mx3005p Real Time PCR system (Agilent Technology, Palo Alto, CA, USA). Relative mRNA levels were examined by the comparative cycle threshold (CT) method and normalized to the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH). We designed and synthesized the primers of pro-apoptotic and anti-apoptotic genes as shown in Table 2.

The extraction of RNA was performed by using the RNeasy Mini Kit (Qiagen) as previously described [21 (link)]. RT-qPCRs for RNA expression were performed by using the KAPA SYBR FAST One-Step RT-qPCR Kit (KAPA biosystems) according to the manufacturer's instructions. RT-qPCRs were performed on Mx 3005p Real-Time PCR System (Stratagene). We designed and synthesized the primers as follows: human GPx-1 (sense, 5′-ctcttcgagaagtgcgaggt-3′; antisense, 5′-tcgatgtcaatggtctggaa-3′), human catalase (sense, 5′-gcagatacctgtgaactgtc-3′; antisense, 5′-gtagaatgtccgcacctgag-3′), human HO-1 (sense, 5′-caggcagagaatgctgag-3′; antisense, 5′-gcttcacatagcgctgca-3′), human Mn-SOD (sense, 5′-gcacattaacgcgcagatca-3′; antisense, 5′-agcctccagcaactctcctt-3′), human CuZn-SOD (sense, 5′-aaggccgtgtgcgtgaa-3′; antisense, 5′-caggtctccaacatgcctct-3′), human GRe (sense, 5′-cagtgggactcacggaagat-3′; antisense, 5′-ttcactgcaacagcaaaacc-3′), and human GAPDH (sense, 5′-cgagatccctccaaaatcaa-3′; antisense, 5′-gtcttctgggtggcagtgat-3′).

Lipopolysaccharide (LPS from Salmonella enterica serotype Typhimurium), indomethacin, and 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Dulbecco’s Modified Eagle Medium (DMEM), 0.25% trypsin-EDTA solution, penicillin/streptomycin (P/S) solution, and fetal bovine serum (FBS) were purchased from Gibco (Grand Island, NY, USA). Dimethyl sulfoxide (DMSO) was purchased from Merck (Darmstadt, Germany). PGE₂ ELISA assay kit was obtained from R&D Systems (Minneapolis, MN, USA). TNF-α ELISA assay kit (ab100747) and nitric oxide assay kit (ab65328) were purchased from Abcam (Waltham, MA, USA). The specific primers were obtained from Integrated DNA Technologies (Coralville, IA, USA). KAPA SYBR FAST One-step RT-qPCR kit was purchased from KAPA biosystems (Wilmington, MA, USA). RNeasy kit was obtained from Qiagen (Hilden, Germany).

Total RNA was extracted from ISE6 cells using All Prep DNA/RNA/PROTEIN Mini Kit (Qiagen, Hilden, Germany) following manufacturer’s recommendations. Gene knockdown levels after TF RNAi were assessed for TF and TG by RT-qPCR on RNA samples using gene-specific oligonucleotide primers (Supplementary Table 1), the Kapa SYBR Fast One-Step RT-qPCR Kit (Kapa Biosystems, Roche Holding AG, Basel, Switzerland), and the QIAGEN Rotor-Gene Real-Time PCR Detection System (Qiagen). A dissociation curve was run at the end of the reaction to ensure that only one amplicon was formed and that the amplicons denatured consistently in the same temperature range for every sample. The mRNA levels were normalized against tick rps4 using the genNorm method [Delta-Delta-Ct (ddCt) method] as described previously (Ayllón et al., 2013 (link)). Normalized Ct values were compared between test siRNAs-treated tick cells and controls treated with Rs86 siRNA by Chi²-test (p = 0.05; n = 4 biological replicates).

One-step PCR including reverse transcription and target amplification was performed using Kapa SYBR FAST One-step RTqPCR Kit (Kapa Biosystems) on a LightCycler 480 qPCR platform with a 96-well format. The specific primers (IDT) for mouse tumor suppressor genes (Hras, 5′-AATTGGGGGAGCAAGGACAT-3′); (Kras, 5′-TATCCTGCTTCCCATCAGTGTTC-3′); (Myc, 5′-GTTGTGCTGGTGAGTGGAGA-3′); (Trp53, 5′-CTTCACTTGGGCCTTCAAAA-3′) and for a housekeeping gene (Gapdh, 5′-CACATTGGGGGTAGGAACAC-3′) were used in the quantitative amplification.
RT-qPCR was initiated by 5 min. and 3 min. incubations at 42°C and 95°C, respectively, followed by 50 cycles (95°C for 10 s, 55°C for 20 s, and 72°C for 20 s) with a fluorescent reading taken at the end of each cycle. Each run was completed with a melting curve analysis (95°C for 5 s, 65°C for 60 s, and 97°C ∞) to confirm the specificity of amplification. Fluorescent values were calculated following the ΔΔCp method on Exor 4 software (Roche (Hungary) Ltd.) and gene expressions are reflected as relative quantification results.