7500 sequence detection system

To further characterize the osteosarcoma lines utilized, we quantified gene expression for BAMBI, SOST, and NOG across 5 standard and 3 xenograft cell lines using quantitative real-time PCR. The experiment was performed both with and without the addition of BMP-2 over a 48 hour period. RNA was extracted using PureLink RNA Mini Kit (Life Technologies, Grand Island, NY) and subsequently converted to cDNA using SuperScript III First- Strand Synthesis System (Life Technologies) according to the manufacturers’ instructions. Gene expression quantitation was carried out using a 7,500 Fast Real-Time PCR system and Taqman Gene Expression assay mix (Life Technologies, Grand Island, NY; Assay IDs: Hs03044164_m1 for BAMBI, Hs00228830_m1 for SOST and Hs00271352_s1 for NOG). The housekeeping gene GAPDH was used as a control and multiple wells of scrambled control were included as negative controls. Reactions for each sample were performed in triplicate. mRNA levels were quantified using ΔΔCt method as per the manufacturer’s instructions, 7500 Sequence Detection System (Life Technologies). MSCs were used as a calibrator.

Total RNA was extracted from fresh tumor and paired normal tissue using Trizol total RNA isolation reagent (Invitrogen) according to the manufacturer’s protocol. miRNA detection was performed using commercial assays (TaqMan MicroRNA assays, Life Technologies) for miR-328 (assay ID: 000543, Life Technologies), in the 7500 Sequence Detection System (Life Technologies). The appropriate negative controls were also run in each reaction. Relative quantification was calculated using the formula 2^−∆∆Ct. Normalization was performed with miR-191.

The number of copies of viral genomes in fcwf-4 cells infected with FCoV was determined using RT-qPCR [11 (link)]. In brief, the medium was removed 20 h after infection, and RNA from the cells was prepared using Isogen (Nippongene), according to the manufacturer’s protocol. Total RNA was reverse transcribed using the PrimeScript RT-PCR Kit (Perfect Real Time; Takara Bio). Viral cDNAs were quantified using qPCR with primers specific for the gene encoding FCoV-N (forward, 5′-TGGCCACACAGGGACAAC-3′; reverse, 5′-AGAACGACCACGTCTTTTGGAA-3′) and the TaqMan probe (FAM-TTCATCTCCCCAGTTGACG-BHQ-1). Reaction mixtures were prepared according to the manufacturer’s protocol using Premix EXTaq (Takara Bio), and sequences were amplified using the 7500 Sequence Detection System (Life Technologies). The viral RNA copy number was normalized to that of the feline gene encoding feline β2-microglobulin (β2M) (GenBank accession no. NM_001009876) using qPCR with forward (5′-CGCGTTTTGTGGTCTTGGTCTTGGT-3′) and reverse (5′-AAACCTGAACCTTTGGAGAATGC-3′) primers specific for β2M. The TaqMan probe, TAMRA-CGGACTGCTCTATCTGTCCCACCTGGA-BHQ-2, was used to detect β2M mRNA.

Total RNA was extracted from the same set of samples using PureLink RNA Mini Kit (Life Technologies, Carlsbad, CA), and subsequently converted to cDNA using SuperScript III First-Strand Synthesis System (Life Technologies, Carlsbad, CA) according to the manufacturers’ instructions. Gene expression quantitation was carried out using a 7500 Fast Real-Time PCR system and Taqman Gene Expression assay mix (Life Technologies, Carlsbad, CA; Assay ID: Hs00181385_m1). The house keeping gene GAPDH was used as an endogenous control normalized to each sample for its mRNA content and multiple wells of scrambled control were included as negative controls. Reactions for each sample were done in triplicate for both IGF1R and GAPDH. Sample mRNA levels were relatively quantified using the ΔΔCT method as described in 7500 Sequence Detection System (Life Technologies, Carlsbad, CA). MSCs were used as the calibrator. Results are reported as the mean plus or minus standard deviation. Student t test was utilized to determine statistical significance between mRNA expression levels in the primary OS samples, primary xenograft samples, and the OS cell lines. P value less than 0.05 was considered statistically significant. Pearson’s r was utilized to determine the correlation between mRNA expression levels in primary OS samples compared with OS cell lines.