Abi 7900 qpcr system

Total RNA was purified from clinical samples and cultured cell lines based on the protocol of TRIzol reagent (Invitrogen). For detection of miR‐133a expression, cDNA was synthesized using One Step Prime script miRNA cDNA Synthesis Kit (Qiagen, Hilden, Germany) following the manufacturer's instructions. Quantitative polymerase chain reaction (qPCR) was conducted using the miScript SYBR Green PCR Kit (Qiagen) under the ABI 7900 qPCR System (Applied Biosystems, Foster, CA). For detection of mRNA expression, cDNAs were synthesized using a reverse transcription kit (Takara, Dalian, China). Quantitative polymerase chain reaction was performed by the SYBR Green Real‐Time PCR Master Mix (Roche, Basel, Switzerland) the manufacturer's instructions. All primes used in this study are listed in Table 2. Glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) and U6 small nuclear RNA were used as internal controls. Relative expression levels were calculated based on 2^−∆∆Ct method from three independent experiments.

RNA was collected from specimens or cultured cells with TRIzol (TaKaRa, Dalian). qPCR was utilized to quantify miRNA, lncRNA and mRNA expression with SYBR kit (Takara, Dalian) on ABI 7900 qPCR system (Applied Biosystems, USA). 2^-ΔΔCt way was used to study relative expression. Primers were utilized as following: ROR1-AS1, 5’-CTGAC GAAAC ACTGG AACTC-3’; 5’-GTCTG ATTTG GTAGC TTGGA TG-3’; GAPDH, 5’-CCAAA ATCAG ATGGG GCAAT GCTGG-3’; 5’-TGATG GCATG GACTG TGGT CATTC A-3’; miR-504 5’-GCTGC TGTTG GGAGA CC-3’; 5’-GCCCT CTGTA TGGGA AAC-3’; U6 5’-CTCGC TTCGG CAGCA CATA-3’; 5’-ACGCT TCACG AATTT GCGT-3’.

Total RNA was extracted with Trizol reagent (Invitrogen, Carlsbad, CA, USA). RNA was reverse transcribed into complementary DNA (cDNA) using PrimeScript Kit (Takara, Shiga, Japan). The real-time quantitative PCR reactions were performed using an ABI 7900 qPCR System (Applied Biosystems, Foster City, CA, USA). Primers used for PCR were as follows: EDIL3 forward: 5′-TGACAGATGGCCGTGGATT-3′, EDIL3 reverse: 5′-TCCTCTTGGCTCCTTGGGTAA-3′; GAPDH forward: 5′-GCACCGTCAAGGCTGAGAAC-3′, GAPDH reverse: 5′-TGGTGAAGACGCCAGTGGA-3′. The conditions of PCR were as follows: 3 min at 95 °C; 40 cycles of 5 s at 95 °C 30 s at 58 °C and 1 min at 65 °C. All qRT-PCR reactions were carried out in duplicate.