Uniq 10 trizol total rna extraction kit

Total RNA was extracted from cells or tissues by using the UNIQ-10/Trizol total RNA extraction kit (Sangon, #B511361, Shanghai, China) and converted to cDNA using the PrimeScript RT Reagent Kit (Takara, #a4302-1, Otus, Shiga, Japan). The SYBR Green PCR kit (Takara, #aka505, Otus, Shiga, Japan) was used for qRT–PCR. The expression levels of gene mRNA were normalized to the GAPDH levels. The following primer sequences were used: ABCB1 (human) sense 5′-CCCATCATTGCAATAGCAGG-3′ and antisense 5′-TGTTCAAACTTCTGCTCCTGA-3′, EGFR (human) sense 5′-TCTACAACCCCACCACGTAC-3′ and antisense 5′-TTCCGTTACACACTTTGCGG-3′, IL6 (human) sense 5′-AGTCCTGATCCAGTTCCTGC-3′ and antisense 5′-AAGCTGCGCAGAATGAGATG-3′, IL16 (human) sense 5′-AAAACATTTTGCGCGCACAA-3′ and antisense 5′-ACCCAGGCACATCATCAGAA-3′, and GAPDH (human) sense 5′-GGTGGTCTCCTCTGACTTCAACA-3′ and antisense 5′-GTTGCTGTAGCCAAATTCGTTGT-3′.
miRNA was isolated using the mirVana^TM miRNA isolation kit (Ambion, #AM1556, Austin, TX). MicroRNA cDNAs were synthesized using the Taqman® MicroRNA Reverse Transcription Kit (Invitrogen, #4366596). Taqman® MiRNA Assays (Invitrogen, #4427975) were used to amplify the expression of miR-338-5p (ID 002658) and RNU6B (ID 001093) according to the manufacturer’s instructions. The amplification and detection of specific products were performed with the Rotor-Gene Q 2plex HRM System (Qiagen, Valencia, CA, USA).

Total RNA was isolated using a UNIQ-10/Trizol total RNA extraction kit (Sangon, Shanghai, China) and was reverse-transcribed into cDNA using the PrimeScript RT Reagent Kit (Takara, Otus, Shiga, Japan). The primer sets used are listed in Supplementary Table 1. Quantitative real-time RT-PCR (qRT-PCR) analysis was performed using SYBR Premix Ex Taq (Takara).
miRNAs were isolated using the mirVana miRNA Isolation Kit (Ambion, Austin, TX) according to the manufacturer's instructions. RT and miRNA detection were conducted using the NCode VILO miRNA cDNA Synthesis Kit and the EXPRESS SYBR GreenER miRNA qRT-PCR Kit, respectively (Invitrogen, Carlsbad, CA, USA). miRNA-specific forward primers were designed according to the manufacturer's instructions; these primers are also listed in Supplementary Table 1. The observed miRNA expression levels were normalized to the U6 expression levels.