C myb sirna

siRNA against c-Myb was synthesized in vitro using a Silencer® siRNA Cocktail Kit (RNase III) (Invitrogen, California, USA) as per the manufacturer’s instruction. Briefly, using c-Myb-specific primers having a T7-promoter leader (8 nucleotides) sequence (Additional file 1: Table S3), a c-myb-specific stretch of cDNA was amplified from LSK-CD34⁻ cells. Amplicons were again amplified using T7 promoter primer. The sense and antisense siRNA templates were transcribed by T7 RNA polymerase and the resulting RNA transcripts were hybridized to create dsRNA. RNAse III was used to produce siRNAs (12–30 base pairs long) which were further purified. The resulting c-myb siRNA or c-jun siRNA (Santa Cruz Biotech, TX, USA) were transfected into sort-purified LSK-CD34⁻ cells using Dharmafect reagent (Thermo Scientific, MA, USA) in a 1:1 ratio. Mock transfected cells were used as controls. Efficiency of silencing of these SiRNA was determined by qRT-PCR using c-Myb- and c-Jun-specific primers. Cells were incubated for 12–18 h and then treated with SNP for 12 h. Levels of c-Jun, c-Myb, and Cd34 mRNA were analyzed by qRT-PCR.