Quantstudio 7 real time pcr system

RNA was isolated using the miRNeasy mini kit (Qiagen). Complementary DNA was synthesized using the Taqman Reverse Transcription Reagents (Life Technologies). Quantitative RT-PCR reactions were performed in duplicates using SYBR Select Master Mix (Life Technologies), respective primers (S1 Table) on a QuantStudio 7 real-time PCR system (Life Technologies). Data was normalized to Hprt endogenous control.
All reagents and kits were used at manufacturer’s recommendation, if not stated otherwise.

RNA extraction from tissue samples was performed using the miRNeasy RNA isolation kit (Qiagen, Valencia, California, USA) and AllPrep DNA/RNA FFPE kit (Qiagen) following the manufacturer’s protocol. The synthesis of complementary DNA (cDNA) was performed from 500 ng of total RNA using the High Capacity cDNA Reverse-Transcription Kit (Invitrogen, Carlsbad, CA, USA). We performed quantitative real-time reverse-transcription analysis (qRT-PCR) using the SensiFAST™ SYBR® Lo-ROX Kit (Bioline, London, UK) and the Quantstudio 7 Real-Time PCR System (Life Technologies, Carlsbad, CA, USA). The average expression levels of the target genes were normalized against β-actin using the comparative CT method. To ensure consistent measurements throughout all assays, for each PCR amplification reaction, three independent RNA samples were loaded on to a plate as internal controls to account for potential plate-to-plate variation, and the results from each plate were normalized against the internal normalization controls. The expression levels of TIAM1 were divided into high and low expression groups according to the median expression values in order to investigate correlation between TIAM1 expression and chemotherapeutic response in the patient cohort.

RNA was isolated using the miRNeasy mini kit (Qiagen; Hilden; Germany). Complementary DNA was reverse transcribed using the Taqman Reverse Transcription Reagents (Life Technologies). Quantitative RT-PCR reactions were performed in triplicates using SYBR Select Master Mix (Life Technologies), respective primers (MWG; Supplementary Table 1) on a QuantStudio 7 real-time PCR system (Life Technologies). Data was normalized to Gapdh endogenous control.
All reagents and kits were used at manufacturer’s recommendation, if not stated otherwise.

For measurement of gene-expression, 2-step PCR was performed. Therefore, cDNA synthesis was conducted by using MMLV Reverse Transcriptase 1st-Strand cDNA Synthesis Kit (Lucigen, Middleton, WI, USA) to synthesize RNA complementary DNA, according to the manufacturer’s instructions. Human-specific primers from the TaqMan Gene Expression Assay System were used to determine gene expression levels (Life Technologies, Carlsbad, CA, USA). Samples were analyzed with the Quant Studio 7 Real-Time PCR System (Life Technologies, Carlsbad, CA, USA). The mean of the human housekeeping genes GAPDH and ribosomal protein L30 (RPL30) was used to normalize human gene expression levels. Specific probes used for gene expression analysis are listed in Supplementary Table S2.

Total RNA was isolated with TRIzol Reagent (invitrogen, 15596026) from tumor cells as Invitrogen user guide. cDNAs were synthesized from 1 µg of total RNA using the SPARKscritpt II RT Plus Kit (SparkJade) and were amplified by 2 × SYBR Green qPCR Mix (SparkJade) using Quantstudio 7 Real-Time PCR System (Life Technologies) according to the manufacturer’s protocols. Relative mRNA expression was evaluated after normalization for Gapdh expression. Each experiment was performed in triplicate. The primers used for quantitative RT-PCR are listed in Supplementary Data 5.

Approximately 100 mg of adipose tissue (gonadal fat pad), 10 mg of liver, and 30 mg of skeletal muscle (triceps) were each processed with a bead homogenizer in Trizol Reagent. RNA was subsequently isolated with RNA isolation kits (Qiagen). cDNA was synthesized from DNase‐treated RNA using the AffinityScript qPCR cDNA Synthesis Kit (Agilent Technologies). SYBR green qPCR was performed using a QuantStudio 7 Real‐Time PCR System (Applied Biosystems) and PowerUp SYBR Green Master Mix (Applied Biosystems). Primers used for determination of relative mRNA levels was produced by Integrated DNA Technologies and are listed in Table 1. All samples were analyzed in triplicate with 18S ribosomal RNA as the control and results are expressed as arbitrary units with calculations by the comparative Ct method.