Rt reaction kit

Total cellular RNA from DMSO and fangchinoline treated SGC7901 cells were extracted after 24 h using TRIzol (Invitrogen) according to the manufacturer’s protocol. One microgram of total RNA was reverse transcribed to cDNA in a total volume of 20 μl system using a RT reaction kit (Promega). Real-time PCR was performed using an Mx 3000P real-time PCR system (Applied Biosystems) according to the manufacturer’s instructions and SYBR Premix Ex Taq (Takara) as a DNA-specific fluorescent dye. PCR was carried out for 50 cycles of 95°C for 10 sec and 60°C for 30 sec. Primer sequences for detection of mRNA expression were synthesized (Table I). All the reactions were repeated at least three times. Gene expression levels were calculated relative to the housekeeping β-actin by using Stratagene Mx 3000P software.

Total RNA of tissues after treating with different factors for 24 h were isolated by TRIzol (Invitrogen, USA) according to the manufacturer's protocol. Complementary DNA was synthesized by reverse transcription of total RNA using a RT reaction kit (Promega, USA). Quantitative real-time PCR was used to determine levels of mRNAs using the Assays-on-Demand Taqman Gene Expression Assays (Applied Biosystems, Foster City, CA, USA) according to the procedure previously described. Results were conducted in triplicate in at least five independent experiments. SYBR Premix Ex Taq (TaKaRa, Japan) was used as a DNA-specific fluorescent dye. Primer sequences were synthesized as shown in table 1.
All the reactions were repeated at least three times. Gene expression levels were calculated relative to GAPDH (glyceraldehyde-3-phosphate dehydrogenase) by using Stratagene Mx 3000P software.

RT-PCR was performed to confirm the knockdown of B7-H6 expression at the mRNA level in HepG2 and SMMC-7721 cells. Total RNA was extracted using TRIzol (Invitrogen) reagent, and purified RNA was then reversely transcribed to cDNA using an RT reaction kit (Promega). RT-PCR was conducted using the ABI 7600 system (Applied Biosystems, USA), and SYBR Green was used as a DNA-specific fluorescent dye. Human GAPDH was selected as a housekeeping gene. Primers were synthesized as follows: GAPDH forward: 5′-TGACTTCAACAGCGACACCCA-3′ and GAPDH reverse: 5′-CACCCTGTTGCTGTAGCCAAA-3′; and human B7-H6 forward: 5′-CTCCTGATTCTGCTGTGGGC-3′ and human B7-H6 reverse: 5′-GTCGGAATGCCTCTTGGTGA-3′. In addition, RT-PCR products were confirmed by electrophoresis on a 1.8% agarose gel containing 0.1% ethidium bromide. Images of the fluorescent bands were captured using a Bio-Rad gel documentation system.

Total RNA of cells treated with or without OMT for 24 h were extracted using TRIzol (Invitrogen) according to the manufacturer’s protocol. One microgram of total RNA was reverse transcribed to cDNA in a total volume of 20 μl system using a RT reaction kit (Promega). Real-time PCR was performed using an Mx 3000P real-time PCR system (Applied Biosystems) according to the manufacturer’s instruction and SYBR Premix Ex Taq (TaKaRa) as a DNA-specific fiuorescent dye. PCR was carried out for 50 cycles of 95°C for 10 s and 60°C for 30 s. Primer sequences for detection of mRNA expression were synthesized as the Table 1.
All the reactions were repeated at least three times. Gene expression levels were calculated relative to the housekeeping β-actin by using Stratagene Mx 3000P software.

Real-time reverse transcriptase-polymerase chain reaction (RT-PCR) was performed to confirm the knockdown of B7-H6 mRNA expression. Total RNA from U87 and U251 cell lines was extracted using TRIzol (Invitrogen) reagent, and was then reverse transcribed into cDNA by using RT reaction kit (Promega). Real-time PCR was performed by using the ABI 7600 system (Applied Biosystems, USA) according to the manufacturer's instruction and SYBR Green was used as a DNA-specific fluorescent dye. Primer sequences for detection of the reference gene, GAPDH and the target gene B7-H6 were synthesized as follows, the human GAPDH, forward primer: 5’-TGACTTCAACAGCGACACCCA, and the reverse primer: 5’-CACCCTGTTGCTGTAGCCAAA-3’; the human B7-H6 forward primer: 5’-CTCCTGATT CTGCTGTGGGC-3’, and reverse primer: 5’-GTCGG AATGCCTCTTGGTGA-3’. The RT-PCR products for B7-H6 and GAPDH genes were also confirmed by using electrophoresis on 1.8% agarose gel containing 0.1% ethidium bromide. Images of the fluorescent bands were captured using Bio-Rad gel documentation system.