Platinum taq polymerase reagents

ChIP analyses were performed using our published procedure [50 (link)]. For RNA isolation and RT-PCR, total cellular RNA was extracted using the TRIzol Reagent kit (Life Technologies, Inc., Rockville, MD). RT-PCR was performed using specific sense and antisense PCR primers. For qRT-PCR detection of miR-34a, miRNA-specific RT-primers (assay IDs: hsa-miR-34a, 000426), TaqMan miRNA Assay (Applied Biosystems, Ambion, Austin, TX) and Platinum Taq Polymerase Reagents (Invitrogen, Grand Island, NY) were used.

For RNA isolation and RT-PCR, total cellular RNA was extracted using the TRIzol Reagent (Life Technologies, Inc., Rockville, MD). RT-PCR was performed using specific sense and antisense PCR primers. Cells were transfected with miR-34a mimic or miR-34a inhibitor or control-miR (Applied Biosystems, Ambion, Austin, TX) using Fugene transfection reagent (Promega Corporation, Madison, WI). Standardization of miR-34a mimic and inhibitor is shown in supplementary figure 4. For qRT-PCR detection of miR-34a, miRNA-specific RT-primers (assay IDs: hsa-miR-34a, 000426), TaqMan miRNA Assay (Applied Biosystems, Ambion, Austin, TX) and Platinum Taq Polymerase Reagents (Invitrogen, Grand Island, NY) were used. Data were calculated by using the standard ΔΔCt method and microRNA expression was represented as fold-difference of each treatment vs. vehicle-treated control. Statistics was performed by using one-way ANOVA and Student's t-test post-hoc analysis. Statistical significance was accepted when p was <0.05. pcDNA3-Flag-LKB1-wild-type (LKB1-WT), Flag-SIRT1, pcDNA3.1-Flag-SIRT3 plasmid constructs were transfected using Fugene 6 (Promega Corporation, Madison, WI) transfection reagent.