Megaplex pools kit

Total RNA was extracted from FFPE tissues using the miRNeasy FFPE Kit (Qiagen, Valencia, CA, USA). The TaqMan^® Array Human MicroRNA A + B Cards Set v3.0 (Applied Biosystems, Foster City, CA, USA) was used for simultaneous measurement of the expression of 760 miRNAs on a microfluidic PCR platform. In brief, 1 μg of total RNA was reverse transcribed using the Megaplex Pools Kit (Applied Biosystems), following which miRNAs were amplified and detected by PCR with specific primers and TaqMan probes. PCR was run in the 7900HT Fast Real-Time PCR system (Applied Biosystems), and SDS 2.2.2 software (Applied Biosystems) was used for comparative analysis of the cycle threshold (ΔC_T). U6 snRNA (RNU6B; Applied Biosystems) served as an endogenous control. ΔC_T was calculated by subtracting the C_T values of U6 from the C_T values of the gene of interest. Expression of each miRNA in the tumor samples was calculated using the equation 2^−ΔCT, where ΔC_T = (C_T miRNA − C_T U6).

The total RNA from the cells stably transfected with DNMT1-silencing vectors or control vectors was extracted with Trizol reagent solution (Ambion) to quantify microRNAs. Expression of miRNAs was analyzed using a TaqMan MicroRNA array (Life Technologies). Briefly, 1 mg of total RNA was reverse-transcribed using a Megaplex Pools kit (Applied Biosystems, Foster City, CA, USA), after which miRNAs were amplified and detected using polymerase chain reaction (PCR) with specific primers and TaqMan probes. U6 was used as an endogenous control. The gene expression is expressed as the ratio of expression relative to that in the control.