Miscript sybr green pcr assay kit

qRT-PCR and Immunoblot was performed as described in [20 (link)]. Briefly For mRNA expression analyses, total RNA was extracted with TRIzol (Invitrogen) and purified using RNAeasy mini columns (Qiagen), and cDNA was generated using M-MuLV first-strand cDNA synthesis kit (New England Biolabs) as per manufacturer’s instructions. Quantitative RT-PCR was performed using Power SYBR-green kit (Applied Biosystems) for mRNA expression analysis or the miScript SYBR-green PCR assay kit (Qiagen), as per manufacturer’s instructions. Actin was used as an internal control. For Immunoblotting whole cell protein extracts were prepared using IP lysis buffer (Pierce) containing Protease Inhibitor Cocktail and Phosphatase Inhibitor Cocktail (Sigma-Aldrich, St.Louis, MO). Protein concentration was estimated using a Bradford Assay kit (Bio-Rad). Proteins separated on 10% or 12% polyacrylamide gels were transferred to PVDF membranes using a wet transfer apparatus from Biorad. Membranes were blocked with 5% skim milk and probed with primary antibodies followed by the appropriate secondary HRP-conjugated antibody (GE healthcare, UK). Blots were developed using the Supersignal Pico Reagent. Antibody and primer information is provided in Table 1.

For mRNA expression analyses, total RNA was extracted with TRIzol (Invitrogen) and purified using RNAeasy mini columns (Qiagen), and cDNA was generated using M-MuLV first-strand cDNA synthesis kit (New England Biolabs) as per manufacturer’s instructions. For miR-146a expression analyses, total RNA was prepared using TRIzol (Life Technologies, Grand Island, NY), small RNAs were enriched using the miRVana kit (Ambion), and cDNA was prepared using the miScript Reverse Transcription Kit (Qiagen) as per manufacturer’s instructions. Quantitative RT-PCR was performed using Power SYBR-green kit (Applied Biosystems, Foster City, CA) for mRNA expression analysis or the miScript SYBR-green PCR assay kit (Qiagen), as per manufacturer’s instructions. GAPDH was used as an internal control. ChIP experiments were performed as described previously (Raha et al., 2005 (link)). MYC binding to the miR-146a promoter and a negative control region was determined using the primers listed in Supplementary file 1E.