Faststart dna master sybr green 1 reaction mix

RNAs were isolated from the murine (BR-5, BR-Luc) and human (IGROV1, SKOV3, A2780 E-80, and A2780) EOC cell lines using TRIzol reagent (Life Technologies). cDNAs were synthesized with random hexamers and MuLV reverse transcriptase (including RNase inhibitor; Applied Biosystems), and purified using a QIAquick PCR Purification Kit (Qiagen).
Human cDNAs were purchased from Origene (HORT502) containing 48 lyophilized cDNAs from EOC patient specimens (8 stage I, 9 stage II, 17 stage III, and 6 stage IV) and 8 cDNAs from normal ovaries. Patient pathology characteristics are in Supplemental Table S1.
Quantitative real-time RT-PCR was performed using a Roche LightCycler 480 real-time PCR (Roche Diagnostics) with gene-specific primers for mouse PCFT, RFC, GARFTase, AICARFTase, and FRα, or human GARFTase and AICARFTase, as appropriate (Supplemental Table S2), and FastStart DNA Master SYBR Green I Reaction Mix (Roche Diagnostics, Indianapolis, IN). Transcript levels were normalized to β-actin transcripts. For the murine transcripts, levels were normalized to transcript levels in mouse liver.

Patient cDNAs were purchased from Origene (Rockville, MD), including 26 non-squamous non-small cell lung cancer (NS-NSCLC) specimens (8, stage I; 5, stage II; 7, stage III; and 6, stage IV) and eight unmatched normal lung specimens. Quantitative real-time RT-PCR was performed using a Roche LightCycler 480 (Roche Diagnostics, Indianapolis, IN) with gene-specific primers and FastStart DNA Master SYBR Green I Reaction Mix (Roche Diagnostics). Transcript levels were normalized to transcript levels of β-actin.