Nanodrop 2000c ultraviolet spectrophotometer

The DS-88 electronic scale was purchased from Wuhan Automation Instrument Factory (Wuhan, China). The high-speed refrigerated centrifuge was purchased from Eppendorf AG (Hamburg, Germany) and Beckman Coulter, Inc. (Brea, CA, USA). The SHH WZI600S digital display - three electric thermostatic water bath box was purchased from the Shanghai Medical Equipment factory (Shanghai, China). The Versa Max MR96A enzyme mark instrument was purchased from Shenzhen Mindray Bio-Medical Electronics Co., Ltd. (Shenzhen, China). NanoDrop 2000c ultraviolet spectrophotometer was purchased from Thermo Fisher Scientific, Inc. (Wilmington, DE, USA). The inverted microscope was purchased from Olympus (Tokyo, Japan) and the light microscope was purchased from Carl Zeiss AG A2 (Oberkochen, Germany).

TRIzol^® (Invitrogen; Thermo Fisher Scientific, Inc.) was used to cleave 786-O, ACHN, 769-P and Caki-1 (all ATCC) cells and liver tissue, and total RNA was extracted by chloroform and precipitated with isopropanol. The RNA concentration was determined by Nanodrop 2000c ultraviolet spectrophotometer (Thermo Fisher Scientific, Inc.). According to the instructions of PrimeScript RT reagent with gDNA Eraser kit (Takara Biotechnology Co., Ltd.), 1 µg RNA was reverse-transcribed into cDNA. An RT-qPCR reaction system was set up according to the instructions of SYBR Premix Ex Taq kit (Takara Biotechnology Co., Ltd.), and iQ5 (Bio-Rad Laboratories, Inc.) was used to assess the expression of HMMR in RCC tissue and cells.
The forward primer for HMMR was 5′-CTGAGAGTGTCTTGGGAG-3′ and the reverse primer was 5′-CAGTGGGTGAGTGACTCTG-3′. GAPDH was used as an internal reference. The forward primer for GAPDH was 5′-CCACTCCTCCACCTTTGACG-3′, and the reverse primer was 5′-TGGTGGTCCAGGGGTCTTA-3′. The cycling program was composed of an initial step to activate the enzyme at 95°C for 3 min, followed by 40 cycles of 95°C for 10 sec, 60°C for 20 sec and 72°C for 1 sec. The 2^−ΔΔCq method (20 (link)) was used to calculate the expression of the genes.