Dnase 1 digestion kit

The messenger RNA (mRNA) and miRNA were isolated from cell samples using RNeasy mini kit (Qiagen) according to the manufacturer’s instructions. Genomic DNA was removed and cDNA was synthesized using DNase I digestion kit (Qiagen) and miScript II RT kit (Qiagen), respectively. Transcripts were amplified using specific primer sets (Supplementary Table S1) and SYBR green PCR kit (Qiagen) with the ABI7500 (Applied Biosystems). Reactions were run in triplicates for each sample and no-template blanks were used as negative controls. Values were normalized to the Gapdh (for mRNA) and U6 snRNA (for miRNA).

Total RNA was extracted from frozen cell pellets using the RNeasy mini RNA extraction kit (Qiagen, Germany) according to the manufacturer’s instructions. Contaminating genomic (gDNA) was removed using on-column DNaseI digestion performed using the DNaseI digestion kit (Qiagen, Germany). Elution of total RNA was performed using 50 μl of DNase/RNase-free H₂O, and quantified with a NanoDrop 2000 UV–vis spectrophotometer (Thermo Scientific, USA).
Total RNA (4 μg) was used as a template for reverse transcriptase reactions, which were carried out in parallel with M-MuLV Reverse Transcriptase (Sangon Biotech, Shanghai, China), following the manufacturer’s instructions. Briefly, total RNA was mixed with 10 μM of random hexanucleotide primers, incubated for 5 min at 70 °C, and kept on ice for 2 min to allow hybridization. Then, RT reaction Mix (buffer 5X, 10 mM each dNTP, RNase inhibitor (20 U/μL)) and reverse transcriptase were added according to the manufacturer’s instructions. After 60 min of incubation at 42 °C, the RT enzyme was heat-inactivated at 70 °C. In each case, the total reaction volume was 20 μL.