Cfx96 touchtm real time pcr detection system

Total RNA was isolated using the same procedure as for RNA-seq analysis. Total RNA (1 μg) was reverse transcribed using random hexamer primers following the manufacturer’s protocol for the QuantiTect Reverse Transcription Kit (Qiagen GmbH, Hilden, Germany). Quantitative RT-PCR was performed in triplicate using the Bio-Rad CFX96 Touch^TM Real-Time PCR Detection System (Bio-Rad, Hercules, CA, United States) with the primers listed in Supplementary Figure S5, according to the QuantiTect SYBR Green PCR Kit manual (Qiagen).
Real-time PCR cycling conditions were as follows: 5 min denaturation at 95°C and 40 cycles of amplification (15 s at 95°C, 30 s at 58°C, and 30 s at 72°C). Relative expression levels were calculated using the expression of actin 2 as an internal reference, according to the ΔΔCt method (Livak and Schmittgen, 2001 (link)). Significant differences in expression in comparison to the control were revealed using the REST tool (Pfaffl et al., 2002 (link)). Product melting curves were generated following PCR to ensure purity of the amplification products. A list of all primers used is included in the supplementary materials (Supplementary Figure S5).

Real-time PCR was performed as described previously (Schmittgen and Livak, 2008 (link)). Total RNA of colon tissue was reverse transcribed to cDNA and subjected to quantitative PCR, which was performed with the BioRad CFX96 TouchTM Real-Time PCR Detection System (BioRad, CA, United States) using iQTM SYBR^® Green Supermix (BioRad, CA, United States), and threshold cycle numbers were obtained using BioRad CFX Manager software. The program for amplification was 1 cycle of 95°C for 2 min followed by 40 cycles of 95°C for 10 s, 60°C for 30 s, and 95°C for 10 s. The relative gene expression was the comparative C_T method. The primer sequences used in this study were as follows:

RNA was extracted from cells using TRIzol (Invitrogen) following the supplied protocol. Final RNA pellet was re-suspended in RNAse-nuclease free water. RNA concentration was determined using Nanodrop 2000c. 500 ng of RNA was subsequently converted to cDNA using the High-Capacity RNA-to-cDNA^™ Kit (Applied Biosystems) following the supplied protocol. cDNA was then used for quantitative real-time PCR using the human Taqman assay for KRAS (Hs00364284_g1), with primer limited GAPDH (Hs02786624_g1) as the internal control and TaqMan^® Universal Master Mix II, with UNG (Life Technologies). Real time PCR was run on the BioRad CFX96 Touch^tm Real-Time PCR Detection System (BioRad) following the standard real time PCR conditions suggested for Taqman assays. Results of all expression levels were calculated as the 2^-ΔΔCt and compared to untreated control cells [26 (link)].

Total RNA was isolated from cell lines with Trizol and reverse transcribed using AMV-RT (Promega, Milan, Italy). After determination of the primer specificity and efficiency, Real-time RT-PCRs were performed with iQTM SYBR^® Green Supermix (Bio-Rad, Milan, Italy) and BIORAD CFX96 TouchTM Real Time PCR Detection System. The oligonucleotides used are listed in Table 1.
The specificity of the Real-time RT-PCR reactions was determined by analyzing the melting curve of the amplified products, which were evaluated by agarose gel electrophoresis, and the 2^−ΔΔCt method could be applied for the analyses.

Total RNA was extracted from tissues or cells using Trizol reagent (Invitrogen) following the supplier’s protocol and first-strand cDNA was synthesized using M-MLV Reverse Transcriptase RNase H Minus (Promega). Relative quantitative Real-Time PCR was carried out on BIO-RAD CFX96 Touch ^TM Real-Time PCR Detection System (BIO RAD) using Bestar ^TM Real-Time PCR Master Mix (SYBR Green methods, DBI Bioscience) with 2^-△△CT method. Actin was used as the reference gene. All primer sequences are listed in Table 1.

qPCR was performed as previously described^{30 (link)}. Total RNA were extracted from cells or tissues and reverse transcribed to cDNA and subjected to qPCR, which was performed with the BioRad CFX96 Touch^TM Real-Time PCR Detection System (BioRad, CA) and iQ SYBR Green Supermix (Bio-Rad, Cat # 1708882), and threshold cycle numbers were obtained using the BioRad CFX manager software version 5.0. The program for amplification was 1 cycle of 95 °C for 2 min followed by 40 cycles of 95 °C for 15 s and 60 °C for 30 s. The primer sequences used in this study are listed in Supplementary Table 1.

Thermal shift experiments were carried out using Sypro Orange dye and a CFX96 Touch^TM Real-Time PCR Detection System (Bio-Rad)^{69 (link)}. A 96-well white PCR plate was used, in which each well contained 20 µL of 2.5 µM wt or R132H IDH1 with 5× Sypro Orange. The standard assay buffer contains 50 mM Tris-HCl at pH 7.5. Various concentrations of substrates were mixed with IDH1 for thermal stabilisation studies. The samples in triplicates were subjected to temperature increases from 20 to 95 °C at 0.2 °C min⁻¹. Bio-Rad CFX Manager was used to determine the melting temperature, T_m.