Revertaid kit

TOP2A, TYMS, and TUBB3 were evaluated with the previously described [22 ] quantitative real-time reverse transcriptase PCR (RT-qPCR) with TaqMan technology on a RotorGene-6000 amplifier (Corbett Research, Mortlake, NSW, Australia). To obtain cDNA on an RNA template, a reverse transcription reaction was performed with a Revert Aid ™ kit (Thermo Fisher, Waltham, MA, USA) with random hexanucleotide primers.

Leaf samples were collected at 4, 48, and 120 h for protein and total RNA isolation. Total RNA was extracted from each sample using TRIzol Reagent Kit (AmbionTM) according to the manufacturer’s instructions. Next, each of the RNA samples was treated with RNase-free DNase (Takara, Dalian, China). Complementary DNA (cDNA) was retro-transcribed from 2 μg of total RNA using the Thermo Scientific RevertAid Kit according to the manufacturer’s instructions. Quantitative real-time polymerase chain reaction (qPCR) was performed on a Bio-Rad real-time detection system (Bio-Rad). PCR conditions were 95 °C for 1 min, followed by 44 cycles at 95 °C, 12 s, 60 °C, 30 s, and 72 °C, 30 s. After cycling, melting curves of the reaction were run from 55 °C to 95 °C. Each reaction was performed in three technical replicates, and the expression profiles of 8 genes were analyzed, with potato ef1a gene used as the constitutive gene for normalization. The quantification of gene expression levels was calculated relative to ef1a with the 2^−ΔΔCT method [87 (link)]. Primer sets used for qRT-PCR are reported in Table S8.

RNA was isolated from 130 tumor samples before treatment using an RNeasy mini kit Plus containing DNase I (Qiagen, Germany) and Ribolock RNAse inhibitor (Thermo scientific, Waltham, MA, USA). The concentration and purity of RNA isolation were assessed with a NanoDrop-2000 spectrophotometer (Thermo Scientific, Waltham, MA, USA; 56–120 ng/μL; A₂₆₀/A₂₈₀ = 1.75–1.85; A₂₆₀/A₂₃₀ = 1.75–2.00). The RIN (RNA integrity number) was 6.4–7.9. The algorithm for assigning RNA integrity values was detailed by Schroeder A. et al. in 2006 [31 (link)]. To obtain cDNA on an RNA template, a reverse transcription reaction was performed using a RevertAid ™ kit (Thermo scientific, Waltham, MA, USA) with random hexanucleotides.

Heart RNA was prepared from RNALater-stored samples using Trizol reagent (Thermo Fisher Scientific, Waltham, MA, USA), glycogen was added according to instructions to visualize the RNA pellet. Liver and spleen RNA was prepared using RNEasy Plus Mini Kit from Qiagen (Düsseldorf, Germany). Homogenization of all samples was performed using Roche Green Bead tubes, using 1 mL of the respective reagent (Trizol or RNEasy Plus buffer plus mercaptoethanol). Samples were reverse-transcribed using RevertAid kit (Thermo Fisher Scientific, Waltham, MA, USA). Real-time PCR was performed on Biorad IQ5 instrument using Biorad IQ SYBR Green mix; primer sequences are given in Table S1. ΔCT values were calculated by subtracting the CT of the gene of interest from the CT of the reference gene; lower ΔCT in graphs means lower abundance of the target cDNA.

At least 1.5 Million cells growing in T75 culture flasks were deprived of medium and treated with 1 ml of TRIZol (Thermofisher). Further workup occurred as per manufacturer description, using 400 µl of chloroform (Alfa Aesar, Molecular Biology Reagent, stabilized with 50 ppm amylene), Iso-Propanol (Sigma, HPLC grade) and Ethanol (Roth, p.A.). The resulting RNA was characterized with a colibri-nanodrop device from Titertek Berthold and used for cDNA synthesis using the RevertAid kit (Thermofisher). For the Q-PCR runs on a StepOnePlus instrument from Applied Biosystems, 100 ng of the resulting cDNA were used per sample using vinculin as a housekeeping gene. The resulting probe volume was 25 µl, using the Quantitect primer system from QIAGEN and evaGreen 5x Q-PCR Mastermix with ROX dye from Solis Biodyne. PCR plates were from Saarstedt. The sample data was evaluated according to the Ct method with the StepOne Software version 2.3.