Lookout dna erase

Trizol reagent (Invitrogen, USA) was used to extract total RNAs. RNA precipitation and washing were performed using 85% ethanol to retain miRNAs. Genomic DNA in RNA samples was removed using LookOut^® DNA Erase (Sigma-Aldrich, USA). The SSRT IV system (Invitrogen, USA) was used to perform reverse transcriptions (RTs) with poly (T) as primer. The qPCR mixtures were prepared using Brilliant III Ultra-Fast SYBR Green QPCR Master Mix (Agilent, USA). Expression levels of GAS5 were normalized to GAPDH endogenous control. For the quantification of miR-21, All-in-One^TM miRNA qRT-PCR Detection Kit (GeneCopoeia, Shanghai, China) was used to perform polyadenylation, RTs and qPCR assays. Expression levels of miR-21 were normalized to U6 endogenous control. Primer sequences were: 5ʹ-ACACTCCAGCTGGGTAGCTTATCAG-3ʹ (forward) and 5ʹ-TGGTGTCGTGGAGTCG-3ʹ (reverse) for miR-21; 5ʹ-CTTCTGGGCTCAAGTGATCCT-3ʹ (forward) and 5ʹ-TTGTGCCATGAGACTCCATCAG-3ʹ (reverse) for miR-21; 5ʹ-AAGGTGAAGGTCGGAGTCAA-3ʹ (forward) and 5ʹ-ATGAAGGGGTCATTGATGG-3ʹ (reverse) for GAPDH; 5ʹ-GCTTCGGCAGCACATATACTAAAATTGGA-3ʹ (forward) and 5ʹ-CTTCACGAATTTGCGTGTCATCCTTG-3ʹ (reverse) for U6. Three replications were set for each experiment. PCR reaction conditions were: 95°C for 1 min, followed by 40 cycles of 9 5°C for 10 s and 55°C for 45 s. 2^−ΔΔCt method was used to calculate the fold changes of gene expression across samples.

HigherPurity™ Total RNA Extraction Kit (Canvax Biotech) was used to extract total RNAs from tissue samples and UM-SCC-17A cells. All RNA samples were digested with LookOut® DNA Erase (Sigma-Aldrich). RNA concentrations were measured by NanoDrop 2000c Spectrophotometer (Thermo Scientific). Tetro Reverse Transcriptase (Bioline) was used to reverse transcribe total RNAs into cDNA, followed by preparation of qPCR mixtures using TaqMan probes and performed following the instructions from TaqMan™ MicroRNA Assay (Thermo Fisher Scientific). GAPDH was used as the endogenous control to measure the expression levels of IUR and p53 mRNA.
High Pure miRNA Isolation Kit (Sigma-Aldrich) was used to extract miRNAs from aforementioned tissue samples and cells. Measurement of the expression levels of mature miR-24 was detected using Custom TaqMan™ Small RNA Assay (Thermo Fisher Scientific). The endogenous control of miR-24 was U6. Fold changes of gene expression were calculated using 2^−ΔΔCT method. All PCR reactions were repeated 3 times.