Steponeplus 96 well real time pcr system

Quantitative real-time polymerase chain reaction (RT-PCR) was used to assess gene expression. RT-PCR reactions were performed in triplicate using a StepOnePlus™ (96-well) Real-Time PCR System (Applied Biosystems) with a total volume of 20 µl. The reaction mixture included 1.5 µl of cDNA, 10 µl of SYBR® Green Real-Time PCR Master Mix, 0.4 µl of ROX reference dye (Takara, Japan), 6.1 µl of dH₂O, and 300 nM of both forward and reverse primers.
The beta-2 microglobulin (B2M) gene was used as the housekeeping gene to normalize the quantity of target genes. StepOneTM software version 2.3 (Applied Biosystems) is used for data analysis after every run. For the final calculation of relative quantity (RQ), we calculated the ratio of the normalized quantity of the treated samples to the normalized quantity of the control sample.
The BRCA1, BRCA2, Bcl2, Bax, TP53, and KRAS genes were considered as target genes. The primers for these genes were designed and synthesized as exon junctions with a length of 18–30 bp, 40%–60% CG content, and a melting temperature of 55–60°C. For this survey, the primers were provided by Metabion (Martinsried, Germany).

Total RNA was extracted using Pure Link™ RNA Mini Kit (Invitrogen, Carlsbad, CA) and quantified using UV-Vis spectrophotometer (Nano Drop 2000, Thermo Fisher). Total RNA was then converted to cDNA by using Verso cDNA synthesis kit (Thermo Scientific, USA). The standard reaction contained 1x Power SYBR Green PCR master mix (Applied Biosystems), primers pairs (Table 3), and cDNA as template strand. The temperature program for 40 cycles was set to denaturation at 94°C for 1 minute and annealing at 55°C for 30 sec. The reaction was conducted on Step One Plus 96-Well Real-Time PCR System (Applied Biosystems). The samples were analysed in triplicate and recA was used as endogenous control for normalization [25 (link)].

Quantification by qRT-PCR was done on the StepOnePlus 96-well real-time PCR system (Applied Biosystems, catalog # 4376600) using the PowerUp SYBR green qPCR kit (Applied Biosystems, catalog # A25918). Two microliters of reverse transcription product were amplified using 500 nM each of the HNqPCRrev1 and HNqPCRfrw1 primers in a 10 μl reaction. A seven-point standard curve of the linear HNDNA or PESDNA was included in every qRT-PCR experiment and used for RNA copy number calculations under the assumption that every RNA molecule was reverse-transcribed into a DNA molecule. Titrations of C. elegans nuclear extract were performed using the in vitro transcription assay followed by qRT-PCR quantification. The DNA products were converted to RNA copy numbers using the standard curve of the linear DNA and then fitted with the Michaelis-Menten model and the non-linear least-squares method in Excel spreadsheets, as described previously [22 (link)]. The resulting fitting equation was used for calculating the maximum yield of RNA copy number and the amount of nuclear extract needed to reach 50% of the maximum yield.

cDNA was synthesized from 1 μg total RNA using Random Hexamers and MuLV Reverse Transcriptase (Applied Biosystems). mRNA expression was analyzed using Platinum SYBR Green qPCR SuperMix (Applied Biosystems) on a StepOnePlus™ 96 well Real-Time PCR System (Applied Biosystems) running StepOne software v2.3 (Applied Biosystems). Primers were designed using Primer-BLAST software (https://www.ncbi.nlm.nih.gov/tools/primer-blast/) or obtained from PrimerBank^{79 (link)} (https://pga.mgh.harvard.edu/primerbank/). Primer sequences are listed in Supplementary Table 4. Relative expression of each gene was calculated relative to the housekeeping gene HPRT1 or Hprt1 as 2^−ΔCt.

Total RNA from lung tissue homogenate was isolated using a peqGOLD Total RNA Kit (Peqlab, Erlangen, Germany) according to the manufacturer's instructions. cDNA was synthesized using Random Hexamers and MuLV Reverse Transcriptase (Applied Biosystems, Darmstadt, Germany). mRNA expression of target genes KC (CXCL1), MCP1 (CCL2), MIP1α (CCL3), IL-6, TNF-α, MMP12, TIMP1, F4/80, CXCL13, IFNγ, Foxp3, Rorγt, Gata3, IL-13, SIRT1, p16 and p21 in comparison to housekeeping control hypoxanthine-guanine phosphoribosyltransferase (HPRT)-1 was determined using Platinum SYBR Green qPCR SuperMix (Applied Biosystems) on a StepOnePlus™ 96 well Real-Time PCR System (Applied Biosystems, Carlsbad, CA). Primers used are listed in Table 1. Relative transcript expression of a gene is given as 2^−ΔCT (ΔCt = Ct_target – Ct_reference), relative changes compared to control are 2^−ΔΔCt values (ΔΔCt = ΔCt_treated– ΔCt_control). Primers were generated using Primer-BLAST software.