Taqman universal pcr master mix protocol

Total RNA of each sample was extracted using an Absolutely PureLink™ RNA Mini Kit (Invitrogen Life Technologies, Carlsbad, CA, USA) and subjected to RT by SuperScript™ First-Strand Synthesis System (Invitrogen) for RT-PCR. All cDNA samples were diluted as a working template in TaqMan™ Fast Advanced Master Mix (Applied Biosystems, Waltham, MA, USA), which was carried out on an ABI 7500 Fast Real-Time PCR System (Applied Biosystems). PCR amplification was conducted by using the TaqMan primers and probes (Assay ID: Hs00176973_m1 for PKC α; Assay ID: Hs01923466 for HMGB1; Assay ID: Hs00179504 for RAGE; Assay ID: Hs00765730 for NF-κB) using the TaqMan Universal PCR Master Mix Protocol (Applied Biosystems) according to the manufactures instructions.

Quantitative real-time PCR was performed on an ABI Prism 7000 sequence detection system (Applied Biosystems, Foster City, CA) by using the 5′ nuclease assay, as we have previously described [72 (link)]. Total RNA was extracted with TRIzol reagent (Invitrogen) according to manufacturer's protocol. One microgram of total RNA were then reverse transcribed into cDNA by using High Capacity cDNA Reverse Transcription Kit (Applied Biosystems) according to the manufacturer’s instructions. PCR amplification was performed by using the TaqMan primers and probes (Assay IDs: Hs 00964396_m1 for human RasGRP3; Hs 01076078_m1 for human EGFR; Hs 00609566_m1 for human IGF1R; Hs 01001580_m1 for human HER-2/ERBB2; Hs 01894962_s1 for human LOR; Hs01588974_g1 for human CYCS; Hs 00157205_m1 for humanCTSD ans Hs 01556702 for human PGR) using the TaqMan Universal PCR Master Mix Protocol (Applied Biosystems). The threshold cycle (Ct) of RasGRP3 was determined and normalized to that of human GAPDH (Assay ID: Hs 03929097_g1) to obtain a ΔCt value (Ct_GAPDH-Ct_{appropriate protein}) from each sample. The Q-PCR was performed in triplicate.

Q‐PCR was performed on a Roche LightCycler 480 System (Roche, Basel, Switzerland) using the 5′ nuclease assay.25, 29 Total RNA was isolated using TRIzol (Life Technologies Hungary Ltd.), DNase treatment was performed according to the manufacturer's protocol, and then, 1 µg of total RNA was reverse‐transcribed into cDNA using High‐Capacity cDNA Kit from Life Technologies Hungary Ltd. PCR amplification was performed using the TaqMan^® Gene Expression Assays (assay IDs: Hs00174092_m1 for interleukin [IL]‐1α, Hs00174097_m1 for IL‐1β, Hs00985639_m1 for IL‐6, Hs00174103_m1 for IL‐8, Hs00174128_m1 for tumour necrosis factor [TNF]‐α; Hs00271958_s1 for HCA₂) and the TaqMan universal PCR master mix protocol (Applied Biosystems). As internal controls, transcripts of 18S RNA (assay ID: Hs03928985_g1) or glyceraldehyde 3‐phosphate dehydrogenase (GAPDH; assay ID: Hs99999901_s1) were determined. The amount of the transcripts was normalized to those of the housekeeping gene using the ΔCT method. Finally, when indicated, the relative expression values were further normalized to the ones of the vehicle‐treated or scrambled RNA‐transfected controls (ΔΔCT method).

RNA was extracted as described above and reverse transcribed to cDNA with Maxima First Strand cDNA Synthesis Kit (Thermo Fisher Scientific, Waltham, MA, USA). Quantitative PCR was performed using TaqMan Universal Master Mix and ABI Prism 7500 sequence detection system (Applied Biosystems, Foster City, CA, USA). The PCR cycling parameters were incubation at 50°C for 2 min, incubation at 95°C for 10 min and thereafter 40 cycles of denaturation at 95°C for 15 s and annealing and extension at 60°C for 1 min. Primers and probes for GAPDH and IL‐6 were purchased from Metabion (Martinsried, Germany). Their sequences were optimised according to the manufacturer's guidelines in TaqMan Universal PCR Master Mix Protocol part number 4304449 revision C (Applied Biosystems) and are presented in Table 2. Expressions of GAPDH and IL‐6 were quantified using the standard curve method as described in the Applied Biosystems User Bulletin. The mRNA levels of metallothionein 1 (MT1) subtypes, IL‐1 receptor antagonist and IL‐36 receptor antagonist were determined with TaqMan Gene Expression assays (Thermo Fisher Scientific; Table 3) by using the 2^−ΔΔCt method. When calculating results, the mRNA expression levels were first normalised against GAPDH.

A cotton pad device–Salivette^™ (Sarstedt AG & CO. KG, Nümbrecht, Germany) was used for saliva collection, according to the manufacturer’s instructions. The total DNA from salivary fluid was extracted and purified by using the EasyMag automatized platform (NucliSENS^® easyMag^® bioMérieux, Durham, NC). All DNA samples were deemed suitable for DNA amplification by PCR based on analysis of the internal control. For TTV detection in saliva, TTV-specific probe and primers were performed as previously described by Maggi et al [20 (link)]. A standard curve with known amounts of a synthetic DNA was performed for TTV absolute quantification by real-time polymerase chain reaction as previously described [21 (link)]. The TTV DNA amplification was based on the TaqMan^™ Universal PCR master mix protocol (Thermo Fisher Scientific, Warrington, UK). The data were analyzed using QuantStudio Design & Analysis Software v.1.4.1. The range of detection of TTV in saliva was 1.6 to 7.4 log₁₀ copies per ml. Positive and negative controls for TTV in saliva were obtained from samples stored in the repository at the Virology Laboratory (Institute of the Tropical Medicine of the Medicine School of the São Paulo University, Brazil). The lower limit of detection of the qPCR assay for TTV was 40 copies per ml (1.6 log₁₀ copies/ml).

Q-PCR was performed on Stratagene Mx3005P QPCR System from Agilent Technologies (Santa Clara, California, United States) using the 5′ nuclease assay. Total RNA was isolated using TRIzol from LifeTechnologies (Carlsbad, California, United States), DNase 5 treatment was performed according to the manufacturer’s protocol, and then 1 μg of total RNA were transcribed into cDNA using 15 IU of AMV reverse transcriptase (4368814, Thermo Fisher Scientific, Rockford, IL, United States). PCR amplification was performed using the TaqMan primers and probes and the TaqMan universal PCR master mix protocol (4369016, Thermo Fisher Scientific, Rockford, IL, United States). As internal control transcripts of Peptidylprolyl isomerase—A (PPIA) (Hs99999904_m1, Thermo Fisher Scientific, Rockford, IL, United States) was applied each cases. Piezo1 gene mRNA expression was measured using PIEZO1 primers (Hs00207230_m1, Thermo Fisher Scientific, Rockford, IL, United States). The amount of the transcripts was normalized at first, to the relevant housekeeping gene using the ΔCT method. The final results were then normalized to the expression of the control or scrambled samples (ΔΔCT method).

RT-qPCR was performed on a LightCycler 480 Instrument II (Roche Life Science, Penzburg, Germany) using the 5′ nuclease assay. Total RNA was isolated using TRIzol (Thermo Fisher Scientific), and DNase treatment was performed according to the manufacturer’s protocol. Then, 1 μg of total RNA was reverse-transcribed into cDNA by using a High Capacity cDNA Kit (Thermo Fisher Scientific). PCR amplification was performed by using the TaqMan assays (IDs: Hs00174092_m1 for interleukin [IL]-1α, Hs00174097_m1 for IL-1β, Hs00985639_m1 for IL-6, Hs00174103_m1 for IL-8) and the TaqMan universal PCR master mix protocol (Thermo Fisher Scientific). As internal controls, transcripts of 18S RNA or peptidylprolyl isomerase A (PPIA) were determined (assay IDs: Hs03928905_g1 and Hs99999905_m1, respectively). The amount of the abovementioned transcripts was normalized first to the expression of the internal control gene, then to the expression found in the relevant control samples using the 2^−ΔΔCt method [48 (link)].