Avian myeloblastosis virus reverse transcriptase

Total RNA was extracted from the AML12 cells using TRIzol (Invitrogen; Thermo Fisher Scientific, Inc.) Total RNA was converted into complementary DNA using avian myeloblastosis virus reverse transcriptase (Takara Bio, Inc., Otsu, Japan). The primers (forward, 5′-CCTGGCTAGAACAAGACACC-3′ and reverse, 5′-ATCCGAGACCCATCGAAA.T-3′) were synthesized by Aoke Company (Beijing, China). RT-qPCR was used to quantify the complementary DNA template. Quantitative gene expression analysis was performed using the SYBR® Premix Ex Taq™ GC (RR071Q; Takara Bio, Inc.) and normalized relative to the β-actin mRNA control band. The reaction system included 12.5 µl of SYBR Premix Ex Taq GC, 0.5 µl of forward primer, 0.5 µl of reverse primer, 2 µl of template and 9.5 µl of H₂O. The reactions were incubated in 96-well optical plates for initial denaturation at 95°C for 3 min followed by 35 cycles of denaturation at at 95°C for 15 sec, annealing at 60°C for 30 sec, extension at 72°C for 30 sec then followed by a final extension at 72°C for 10 min.

Total RNA was extracted from cells using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.). RNA samples were qualified by using a UV spectrophotometer, and optical density (OD)₂₆₀/OD₂₈₀ was limited to 1.8–2.0. Total RNA was reverse transcribed to cDNA using a reverse transcription system (cat. no. 4368814; Fermentas, Thermo Fisher Scientific, Inc.). In brief, total RNA was incubated at 70°C for 10 min and centrifuged at 1,000 × g for 20 min at room temperature. cDNA was synthesized by Avian Myeloblastosis Virus Reverse Transcriptase (Takara Bio, Inc., Otsu, Japan), according to the manufacturer's protocols. RT-qPCR with SYBR Green qPCR dye (Toyobo Life Science, Osaka, Japan) was conducted, according to the manufacturer's protocol, to study the quantitative expression of miR-16. The thermocycling conditions were as follows: 45°C for 5 min for reverse transcription, followed by 94°C for 30 sec and then 40 cycles of 94°C for 5 sec and 60°C for 30 sec. Three replicates were included in the RT-qPCR analysis. The relative expression levels of miR-16 were normalized to β-actin and calculated using the 2^−∆∆Cq method (14 (link)). The sequences of the primers used are: miR-16, 5′-TAGCAGCACGTAAATATTGGCG-3′ (forward) and 5′-TGCGTGTCGTGGAGTC-3′ (reverse); and GAPDH, 5′-AGAAGGCTGGGGCTCATTTG-3′ (forward) and 5′-AGGGGCCATCCACAGTCTTC-3′ (reverse).

Total RNA was extracted from cells by Trizol reagent (Thermo Fisher Scientific) then reverse transcribed and synthesized to cDNA using avian myeloblastosis virus reverse transcriptase (Takara, Dalian, People’s Republic of China) according to these manufacturers’ instructions. The quantification of gene transcripts was determined by real-time PCR using SYBR Green Real time PCR Master Mix (Toyobo) and Mx3000P quantitative PCR (qPCR) system (Stratagene, La Jolla, LA, USA). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as the internal control. The qPCR primers are 5′-CTCCAAGAAGCTGGTCATCA-3′ and 5′-GAGCTCCTCGAGGTTGTACC-3′ for CUL4A; 5′-TACACTGCCCAGGAGCCAGA-3′ and 5′-TGGCACCAGTGTCCGGATTA-3′ for E-cadherin; 5′-GACGGTTCGCCATCCAGAC-3′ and 5′-TCGATTGGTTTGACCACGG-3′ for N-cadherin; 5′-TGAGTACCGGAGACAGGTGCAG-3′ and 5′-TAGCAGCTTCAACGGCAAAGTTC-3′ for vimentin; 5′-GCACCGTCAAGGCTGAGAAC-3′ and 5′-TGGTGAAGACGCCAGTGGA-3′ for GAPDH.