Abi 7900ht sequence detection system with sybr green dye

Trizol reagent (Invitrogen, Carlsbad, CA, USA) was used to extract total RNA from the tissue samples according to the manufacturer’s protocol. Superscript II reverse transcriptase and random primers were used to synthesize cDNA (Toyobo, Osaka, Japan). Quantitative real-time PCR (qRT-PCR) was conducted on the ABI 7900HT Sequence Detection System with SYBR-Green dye (Applied Biosystems, Foster City, CA, USA; Toyobo, Osaka, Japan). The reaction parameters included a denaturation program (5 min at 95 °C), followed by an amplification and quantification program over 40 cycles (15 s at 95°C and 45 s at 65°C). Each sample was tested in triplicates, and each sample underwent a melting curve analysis to check for the specificity of amplification. Table 1 illustrates the primer sequences of hub genes. The expression level was determined as a ratio between the hub genes and the internal control GAPDH in the same mRNA sample, and calculated by the comparative CT method. Levels of COL1A2, COL1A1, COL4A1, THBS2 and ITGA5 expression were calculated by the 2^−ΔΔCt method.

Total RNA was extracted from bone marrow samples from AML patients and normal individuals using Trizol reagent (Invitrogen, Carlsbad, CA, U.S.A.) according to the manufacturer's protocol. Superscript II reverse transcriptase and random primers were used to synthesize cDNA. Quantitative real-time PCR (qRT-PCR) was performed on the ABI 7900HT Sequence Detection System with SYBR-Green dye (Applied Biosystems, Foster City, CA, U.S.A.). All primers are listed in Table S2. Expression levels of PTPN2 were calculated using the 2-ΔΔCT method.

Trizol reagent (Invitrogen, Carlsbad, CA, U.S.A.) was used to extract total RNA from the Beas-2B cells and A549 cells according to the manufacturer’s protocol. Superscript II reverse transcriptase and random primers were used to synthesize cDNA. Quantitative real-time PCR (qRT-PCR) was conducted on the ABI 7900HT Sequence Detection System with SYBR-Green dye (Applied Biosystems, Foster City, CA, U.S.A.). All primers were shown in Table 1. The reaction parameters included a denaturation program (10 min at 95°C), followed by an amplification and quantification program over 45 cycles (15 s at 95°C and 34 s at 60°C). Each sample was tested in triplicates, and each sample underwent a melting curve analysis to check for the specificity of amplification. The expression level was determined as a ratio between the hub genes and the internal control GAPDH in the same mRNA sample, and calculated by the comparative CT method. Expression levels of hub genes were calculated by the 2^−δδCt method.