Quantification of GAS5 Expression

TRIzol reagent (Invitrogen) was used for isolating total RNA from SV40 MES-13 cells at different groups according to manufacturer’s instructions [23 (link)]. Then, SuperScript™ IV One-Step RT-PCR System (catalog number: 12594025, Invitrogen) was used for the preparation of cDNA. QuantStudio®3 Quantitative real-time PCR instrument (Applied Biosystems) was used to perform qRT-PCR reactions. A total fluid volume of 25 μl and TaqMan™ Fast Advanced Master Mix (catalog number: 4444556, Applied Biosystems) were used for the amplification. A two-step cycle protocol was used for detecting GAS5 expression. ABI Prism 7000 SDS software (Applied Biosystems) was used for analyzing the data. The relative expression of GAS5 was calculated by comparative method 2^−ΔΔC_t.