Sybr green one step rt pcr master mixes

Total RNA was extracted with the use of TRIZOL reagent (Invitrogen, USA). The synthesis of cDNA was performed with the Revert aid first-strand cDNA synthesis kit (Thermo Fisher, USA). The qRT-PCR was carried out using SYBR Green One-Step RT-PCR Master Mixes (Thermo Fisher) on a Bio-Rad CFX96 Touch qPCR system (Bio-Rad, USA). Primers were supplied by GenePharma (Shanghai, China). The sequences were as follows: ADRA2A (sense): 5′-AGCTCGCTGAACCCTGTTAT-3′ (anti-sense): 5′-CTGACCAGGGTCTGTAAGCA-3′; GAPDH (sense): 5′-GCACCGTCAAGGCTGAGAAC-3′ (anti-sense): 5′-GCCTTCTCCATGGTGGTGAA-3′. The relative gene expression levels were calculated using the 2^−ΔΔCt method.

Total RNA from tissue and cells was extracted with the use of TRIZOL kit (Invitrogen, USA). Reverse-transcribed complementary DNAs (cDNAs) were prepared according to the Revert aid first-strand cDNA synthesis kit (Thermo Fisher, USA), and qRT-PCR was conducted utilizing the SYBR Green One-Step RT-PCR Master Mixes (Thermo Fisher) on an ABI 7900 real-time PCR system (Applied Biosystems, USA). We used U6 and GAPDH as internal control. The primer sequences were as follows: miR-6071 (sense): 5′-TGGTACTGATGTGATGGACT-3′ (anti-sense): 5′-TCATATCACACAGCACCGAT-3′; U6 (sense): 5′-CTCGCTTCGGCAGCACA-3′ (anti-sense): 5′-AACGCTTCACGAATTTGCGT-3′; ULBP2 (sense): 5′-AAATGTCACAACGGCCTG-3′ (anti-sense): 5′-TGAGGGGTTCCTTGGG-3′; GAPDH (sense): 5′-CGAGCCACATCGCTCAGACA-3′ (anti-sense): 5′-GTGGTGAAGACGCCAGTGGA-3′. The relative gene expression levels were calculated using the 2^−ΔΔCt method.