Total cellular RNA (1 to 2 μg in a total volume of 10 μl) was extracted using TRIzol (Life Technologies) following the manufacturer’s protocol and was mixed with 1 μl of 10 mM deoxynucleotide triphosphates (2.5 mM of each; LifeTechnologies) and 1 μl of 50 μM random hexaprimers (New England Biolabs). Purified RNA was treated with deoxyribonuclease I (DNase I) (Takara) and reverse-transcribed using RevertAid reverse transcriptase (Fermentas). Complementary DNA (cDNA) was amplified by an SYBR Green PCR master mix (Applied Biosystems). Quantitative polymerase chain reaction (qPCR) was performed using SureCycler 8800 (Agilent) and AriaMx Real-Time PCR System (Agilent). The reaction contained 5 ng of cDNA, 2 μl of 1 μM qPCR primer pair (final concentration of each primer 200 nM), 5 μl 2× Master Mix, and final volume made up to 10 μl with DNase-free water. The specificity of the reaction was verified by melt curve analysis. Each reaction was performed in three replicates. Fold changes were calculated by the ΔΔCt method using GAPDH or β-actin as internal standards and normalized to the experimental control as indicated. For nucleic acids extraction (total RNA and genomic DNA) from FFPE tumor samples, we used the QIAGEN FFPE All-Prep kit and followed the manufacturer’s protocol. Small portion of specimens were prepared from ~80-μm slices of FFPE tumor blocks, followed by dewaxing using Deparaffinization Solution (QIAGEN). Purified RNA was subjected to reverse transcription PCR (RT-PCR) and qPCR as mentioned above. Primers used in this study are provided in table S2.

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