Quantitative Analysis of miR-494-3p and Bmi1 Expression

Total RNA was extracted by Quick-RNA^TM MiniPrep Kit (Zymo Research Corporation, Irvine, CA, USA). For miR-494-3p detection, 100 ng extracted RNA was used for complementary DNA (cDNA) synthesis with RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific Inc.) and a specific RT primer (RiboBio Co., Ltd.). qPCR was performed on an ABI StepOnePlus™ Real-Time PCR System (Applied Biosystems, Life Technologies Corp., Carlsbad, CA, USA) by SYBR Green reagent (SYBR^® FAST qPCR Kit, Kapa Biosystems, Inc., Wilmington, MA, USA) with specific primers purchased from RiboBio Co., Ltd. and analyzed with the StepOne software (Applied Biosystems). For Bmi1 detection, 1 μg of extracted RNA was used for cDNA synthesis with random hexamers provided in RevertAid First Strand cDNA Synthesis Kit followed by SYBR Green based qPCR reaction. The primer sequences were listed as followed:

Bmi1

Forward: 5′-AATCCCCACCTGATGTGTGT-3′

Reverse: 5′-GCTGGTCTCCAGGTAACGAA-3′

MRPL19 (internal control)

Forward: 5′-GGGATTTGCATTCAGAGATCAG-3′

Reverse: 5′-GGAAGGGCATCTCGTAAG-3′

qPCR data were analyzed as previous described [28 (link)].