Abi 7500 real time pcr thermocycler

Quantitative PCR (qPCR) reactions were performed using PowerUp SYBR Green Master Mix (Applied Biosystems). Each qPCR reaction contained 10 ng of cDNA as template, 300 nM of each primer (Table 2) and 1x of PowerUp SYBR Green Master Mix; in a final volume of 10 µL adjusted with nuclease-free water (Thermo fisher). The PCR program consisted of 2 min 50°C for UDG activation, 2 min 95°C for Dual-Lock DNA polymerase followed by 40 cycles at 95°C for 15 sec and 60°C for 1 min. Primers for each target gene were designed using Primer3 web (Untergasser et al., 2012 (link)). Each qPCR reaction was performed in duplicates with a negative control in each run using an Applied Biosystems ABI 7500 real-time PCR thermocycler (Applied Biosystems). The amplification specificity of each PCR product was monitored using the melting curve analysis in Sequence detection system (SDS) version 1.4.0.27 (Applied Biosystems) and visualizing the PCR products in a 2% agarose gel. The relative gene expression was estimated by the 2^-ΔΔCT method (Livak and Schmittgen, 2001 (link)), using β-actin as a reference gene (GenBank XM_026821238.1) as was previously described (Hajeri et al., 2014 (link)).

The mRNA levels were assessed by real-time polymerase chain reaction (PCR). In brief, the total RNA was extracted from liver, colon, spleen, epididymal white adipose tissue (eWAT) and lung tissue with Trizol according to the manufacturer’s protocol (Life technologies, Carlsbad, CA, USA) and reverse-transcribed using cDNA supermix (QuantaBio, Beverly, MA, USA). Quantitative real-time PCR was performed on an ABI 7500 real-time PCR thermocycler, whereas SYBR green PCR Master Mix (Applied Biosystems, Foster City, CA, USA) was used for real-time PCR analysis. The relative quantities of target transcripts were calculated from duplicate samples after normalization of the data against the housekeeping gene, 18S rRNA (mouse). Relative mRNA expression was calculated using the delta-delta Ct method. The primer pairs used were listed in Table S1.

QRT-PCR was used to validate the results of aCGH. The total RNA was extracted from RMS tissues and 14 cases normal muscle tissues as controls using RNeasy FFPE Kit (QIAGEN), and the total RNAs from all the samples were treated with DNAse I, and transcribed to single-stranded cDNA by reverse transcription using QuantiTect Reverse Transcription Kit (QIAGEN). A house keeping mRNA, ACTB (Hs_ACTB_2_SGQuantiTect Primer Assay, QT01680476), was assessed in all samples. The normal muscle tissues were used as control. The Primers GLI1 (QT00060501) and GEFT (QT00202916) genes were from QuantiTect Primer Assays (QIAGEN). The reaction was carried out on the ABI 7500 Real-Time PCR thermocycler (Applied Biosystems) using Quantifast SYBR Green PCR Kit (QIAGEN). The following thermal cycling program was applied: 5 min at 95°C, 40 cycles of 10 s at 95°C and 30 s at 60°C. Data were normalized for ACTB expression using comparative threshold cycle method. All PCRs were done in triplicates. Cycle threshold (Ct), the fractional cycle number at which the amount of amplified target reached a fixed threshold, was determined. ΔCt values were calculated by subtracting the ACTB Ct values from the target gene Ct values (ΔCt = Ct (GLI1 or GEFT gene in RMS/normal muscle sample) - Ct (ACTB gene in RMS/normal muscle sample)). Expression level was determined as 2-ΔCt.