Rnapure tissue cell kit dnase 1

Total RNA extracted from cells and tissues using the RNAPure Tissue & Cell Kit (DNase I) in accordance with the manufacturer's instructions (CWBIO, Beijing, China). Then, a total of 1 μg RNA was submitted to the reverse transcription of miRNA and mRNA using stem-loop primers and random primers with TaKaRa system (Dalian, China) according to the manufacturer's instructions. Subsequently, the RT-PCR was carried out using a TaqMan Universal Master Mix II kit on a Bio-Rad detection system (Bio-Rad, Hercules, CA). GAPDH and U6 expression levels are served as internal references to normalize mRNA and miRNA expressions, respectively. Primers were listed as follows.
LUCAT1: forward- (F-) 5′-CCTCACAAGAAGCTCACCCA-3′, reverse- (R-) 5′-CAGCATGTAGCCCATGGTAGA-3′; KRAS: F-5′-TAGGCAAGAGTGCCTTGACG-3′, R-5′-CCCTCCCCAGTCCTCATGTA-3′; and GAPDH: F-5′-CCACTAGGCGCTCACTGTTCTC-3′, R-5′-ACTCCGACCTTCACCTTCCC-3′. If the expression was lower than the median value, miR-181c-5p was considered as the low expression and vice versa. Similarly, LUCAT1 was considered to express at a high level when its expression was higher than the median value and vice versa.

The total RNAs were extracted from cells using the RNApure Tissue & Cell Kit (DNase I) in accordance with the manufacturer’s instructions (CWBio, Beijing, China). Then, a total of 1 μg RNA from each sample was subjected to cDNA reverse transcription and real-time PCR (RT-PCR) using the Quant One Step RT-PCR kit (TIANGEN, Beijing, China) on Bio-Rad detection system (Bio-Rad, Hercules, CA) after RNA quantification via using an ND-1000 NanoDrop Spectrophotometer (NanoDrop Technologies, Inc., Wilmington, Delaware). GAPDH expression level serves as an internal reference. Primers were listed as follows,
CD47: forward (F) 5′-CGGCGTGTATACCAATGC-3′; Reverse (R) 5′-TTTGAATGCATTAAGGGGTTCCT-3′;
GAPDH: F 5′-CCACTAGGCGCTCACTGTTCTC-3′; R 5′-ACTCCGACCTTCACCTTCCC-3′.