Nuclear and Cytoplasmic RNA Isolation

Accordingly 29 , 33 (link), cells were collected, resuspended in lysis buffer (10 mM NaCl, 20 mM MgCl, 10 mM Tris-HCl, pH 7.8, 5 mM DTT, 0.5% NP-40) and kept in ice for 5 minutes. Nuclei were pelleted by centrifugation at 8000 rpm for 5 minutes at 4°C, pellets were washed and resuspended in lysis buffer. The cytoplasmic fraction was collected to a new tube and clarified by centrifugation. Nuclear and cytoplasmic fractions were subjected to protease treatment for 20 minutes at 37°C by adding an equal volume of proteinase K solution (300 mM NaCl, 0.2 M Tris-HCl, pH7.5, 25 mM EDTA, 2% SDS and 0.1 mg/ml proteinase K), and RNA was purified using the QIAzol Lysis Reagent (Qiagen, Germany). The RNA was subjected to DNase treatment; cDNA was synthesized using the QuantiTect Reverse Transcription Kit (Qiagen, Germany) according to the manufacture's protocol. Quantitative real time PCR was performed on StepOnePlus real-time PCR machine (Applied Biosystems, UK), using SYBR Green Universal PCR Master Mix (Applied Biosystems, UK). Oligonucleotides used for quantitative and semi-quantitative PCR are listed in Table S3.