Taqman power sybr green pcr mix kit

TRIzol reagent (Invitrogen, Carlsbad, CA) was utilized to separate total RNA in tissues. First-strand complementary DNA (cDNA) was synthesized using 1 μg of total RNA and the PrimeScript RT reagent Kit (Promega, Madison, WI). qRT-PCR was applied to measure the relative RNA level using the TaqMan Power SYBR Green PCR Mix kit (Thermo Fisher Scientific, Waltham, MA). qPCR was performed using Applied Biosystems 7500 Real Time PCR System (Applied Biosystems, Foster City, CA). The thermal cycle was as follows: pre-denaturation at 95°C for 1 min, followed by 40 cycles at 95°C for 15 s, 60°C for 30 s, and 72°C for 30 s. The threshold cycle was determined after the reactions. The relative miRNA and lncRNA expression levels were calculated based on comparative threshold cycle (Ct) technique (2^−ΔΔCt). The expression level was normalized to GAPDH levels of each sample. The primer sequences were as follows: SUMO1P3 primers, forward: 5′-ACTGGGAATGGAGGAAGA-3′, reverse: 5′-TGAGAAAGGATTGAGGGAAAAG-3′; GAPDH primers, forward: 5′-CGCTCTCTGCTCCTCCTGTTC-3′, reverse: 5′-ATCCGTTGACTCCGACCTTCAC-3′.

Total RNA was extracted from the HCC tissues and HCC cell lines by TRIzol reagent (Takara Bio, Otsu, Japan) following the manufacturer’s instructions. First-strand complementary DNA (cDNA) was synthesized using 1 μg of total RNA and the PrimeScript RT reagent Kit (Promega, Madison, WI, USA). qRT-PCR was applied to measure the relative RNA level using the TaqMan Power SYBR Green PCR Mix kit (Thermo Fisher Scientific, Waltham, MA, USA). qPCR was performed using Applied Biosystems 7500 Real Time PCR System (Applied Biosystems, Foster City, CA, USA), under the following conditions: 10 min 95°C, followed by 40 cycles of 10 s 95°C, 30 s 60°C, and 30 72°C. The level of RP5‑833A20.1 was normalized to the level of h-actin using the 2^−ΔΔCT method. H-actin, forward, 5ʹ-GTCCACCGCAAATGCTTCTA-3ʹ; reverse, 5ʹ-TGCTGTCACCTTCACCGTTC-3ʹ. RP5‑833A20.1, forward, 5ʹ- TCTCCAACAGGGTGAGGAAAC-3ʹ; reverse, 5ʹ- TCTGAGATGCTGGGTGCAAG-3ʹ.