M1705

To reduce experimental variations, we performed cDNA synthesis for all samples at once. Reverse transcription reaction was performed as described before [57 (link)] using a master mix consisting of 1x M-MLV buffer (M1705, Promega, Walldorf, Germany), 40 nmol dNTP mix (L785.2, Carl Roth, Karlsruhe, Germany), 0.1 nmol oligo-dT18 primer (SO131, Thermo Fisher Scientific, Waltham, MA, USA), 0.1 nmol random hexamer primer (SO142, Thermo Fisher Scientific, Waltham, MA, USA), 40 U RNase inhibitor (EO0381, Thermo Fisher Scientific, Waltham, MA, USA), and 200 U M-MLV reverse transcriptase (M1705, Promega, Walldorf, Germany). Samples were incubated for 1 h at 37 °C followed by inactivation of the transcriptase at 95 °C for 2 min.

HCT116 cell cultures were treated with 5-FU (20 μM) or oxaliplatin (12.5 μM) for 48 h (n = 4/group). After treatment, cultures were processed for RNA extraction with Tri-reagent (TR 118, Medical Research Centre) according to the manufacturer’s instructions. Genomic DNA was removed using the Ambion Turbo DNA-free kit, followed by reverse transcription using an M-MLV reverse transcriptase for 2 h at 37 °C (M1705, Promega). Quantitative real time PCR was carried out in technical triplicates on a Light Cycler 480 (Roche) using the SYBR select master mix (4472918, Life Technologies) according to manufacturer’s instructions. Reactions for each target were optimised for an efficiency >1.8 and specificity was confirmed via melt-curve analysis. The following primers (KiCqStart SYBR green, Sigma) were used: translationally controlled tumour protein (TCTP/TPT1) (sense, 5’-TACTCTTTCTGGTCTCTGTTC-3’; antisense, 5’-CAAGTTTCACAAAAGAAGCC-3’), and β2-tubulin (B2T) (sense, 5’-AAGGACTGGTCTTTCTATCTC-3’; antisense, 5’-GATCCCACTTAACTATCTTGG-3’) at 400 nM in 20 μL reactions with 80 ng cDNA. Treatment effects on TCTP mRNA expression were assessed using one-way ANOVA, followed by Tukeys post-hoc analysis, where applicable. Analyses were performed using SPSS 19.0 (IBM). Statistical significance was accepted at P < 0.05 and data are presented as the mean ± standard error of the mean (SEM).

For reverse transcription, equal concentrations of RNA were diluted with nuclease-free water (T143.5, Carl Roth, Karlsruhe, Germany) to a volume of 5.5 µL. To each sample, 2 µL of M-MLV reverse transcriptase buffer (M1705, Promega, Madison, WI, USA), 0.5 µL of OligodT (SO132, Thermo Fisher Scientific, Waltham, MA, USA), 0.5 µL of random hexamer primer (SO142, Thermo Fisher Scientific, Waltham, MA, USA), 0.5 µL of dNTPs (L785. 2, Carl Roth, Karlsruhe, Germany), 0.5 µL RNAse inhibitor (EO0382, Thermo Fisher Scientific, Waltham, MA, USA), and 0.5 µL reverse transcriptase (M1705, Promega, Madison, WI, USA) were added. To avoid errors due to pipetting, the mixture was prepared as a master mix for the entire experimental approach, so 4.5 µL of the master mix was added to 5.5 µL of the adjusted RNA sample. After mixing, samples were incubated at 37 °C for 1 h, followed by inactivation at 95 °C heat for 2 min and stored at 4 °C.

Total RNA was extracted from frozen tumor specimens utilizing Trizol (93289‐100ML, Sigma‐Aldrich; Merck KGaA) and cDNA was synthesized by reverse transcription (M1705, Promega, USA) in the reaction system of 25 μL. q‐PCR was performed in duplicate for each target gene using SYBR Green dye (Promega, USA) as the fluorophore. The analysis was carried out on an ABI7500 Detection System (Applied Biosystems). Gene expression levels were quantified employing the 2^‐ΔΔCt method, with GAPDH as the reference gene for normalization. The primer sequences for the target genes were custom synthesized by Genecreate Biotechnology Co., Ltd. A comprehensive list of the primer sequences can be found in Table 1.

RNA was extracted from INS-1 832/13 cells with TRIzol (Invitrogen #15596026) and 500 ng RNA was used for first-strand cDNA synthesis in a reaction containing 50 ng of random primers (Promega #C1181), 500 μM dNTPs (Fisher Bioreagents #BP2564-1), 1× first-strand buffer, and 200 units of Moloney murine leukemia virus reverse transcriptase (Promega #M1705).