High pure rna isolation kit

A High Pure RNA Isolation Kit (Roche Ltd., Mannheim, Germany) was used to extract total RNAs from the cells according to the manufacturer’s protocol [16,17] . One microgram of total RNAs was reverse-transcripted to produce first strand cDNA using the PrimeScript RT reagent Kit with gDNA Eraser (TaKaRa, Shiga, Japan) according to the manufacturer’s protocol [16,17] . The Thermal Cycler Dice Real Time System (TaKaRa, Shiga, Japan) was used for a 2-step reverse transcription polymerase chain reaction (PCR). The mRNA transcripts were quantified by SYBR Premix ExTaq (TaKaRa, Shiga, Japan). HO-1 specific primers 5′-GCTCAAAAAGATTGCCCAGAA-3′ and 5′-TCACATGGCATAAAGCCCTACA-3′ were used. The other primers were purchased from TaKaRa. The amplification conditions included 30 s at 95 °C, a run of 45 cycles at 95 °C for 5 s, and 60 °C for 60 s followed by dissociation for 15 s at 95 °C and 30 s at 60 °C and then 15 s at 95 °C on the Thermal Cycler Dice Real Time System. The Thermal Cycler Dice Real Time System analysis software (TaKaRa, Shiga, Japan) was used to analyze the data. The Ct values (cycle threshold) were calculated by the crossing-point method, and the relative quantities of target mRNA expression levels were measured by the comparison of standard curve. The results for each sample were normalized to ACTB, the housekeeping gene.

RNA from each condition and the controls was extracted using the High Pure RNA Isolation Kit (Roche, Basel, Switzerland) following the manufacturer’s instructions. RNA was quantified using a nanodrop and 1 µg of RNA was converted to cDNA using the High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific). Relative gene expression was evaluated using 10 ng of cDNA, 250 nM of each primer (Table S1), and by using the Fast SYBR Green Master Mix (Thermo Fisher Scientific) with an annealing temperature set to 60 °C. Melting curves were performed at the end to assess if the primers were amplifying only the correct amplicon. The values were treated following the 2^−ΔΔCT method. GAPDH gene expression was used as an endogenous control and the relative expression was calibrated using day 0 of differentiation.

For RNA sequencing purposes (RNA-seq), three aliquots of the cell line and specimens (tumour core and liver) from seven animals were collected. The RNA purification system PeqGold TriFast (Peqlab, Erlangen, Germany) was used to isolate RNA from metastatic liver tissue. Briefly, specimens were defrosted in PeqGold TriFast (1 ml/100 mg tissue) and then homogenized using TissueLyser LT (Qiagen, Hilden, Germany) at 50 Hz. Total RNA was isolated according to the manufacturer’s instructions and stored at −80 °C. In addition, the High Pure RNA Isolation Kit (Roche, Grenzach-Wyhlen, Germany) was used to isolate RNA from CMT-93 cells according to the manufacturer’s recommendations. The quantity and integrity of the isolated RNA was assessed in a NanoDrop ND − 1000 spectrophotometer, version 3.5.2 (Peqlab, Erlangen, Germany), using the 260 nm/280 nm absorbance ratio and was further analysed with an Agilent 2100 BioAnalyzer (Agilent Technologies, Santa Clara, California, USA) as a quality check. RNA-seq was performed at the Transcriptome and Genome Analysis Laboratory in Goettingen, Germany, using an Illumina HiSeq2000 sequencer (Illumina, Inc., San Diego, California, USA).