Reverse transcriptase assays. A. Fluorogenic assay. RT activity was measured by detection of RNA:DNA heteroduplex by fluorescence of EvaGreen binding. Oligo dT primed poly A was incubated at 37°C and 65°C in the presence of indicated Pol enzymes in manufacturer recommended buffers and dTTP. Fluorescence measurements were obtained every 6 seconds for 10 minutes. The initial slopes from a plot of RFU vs. time in seconds were determined by linear least square regression from 30 to 150 seconds at 37°C and from 30 to 90 seconds at 65°C. Error bars are standard error of regression slope. B. RT primer extension assay. HEX-labeled dT 20 primed poly A was incubated 10 minutes at 37°C and then 10 minutes at 65°C in the presence of indicated Pol enzymes and dTTP in manufacturer recommended buffers. Primer extension products were resolved by 10% denaturing PAGE and imaged on a Molecular Imager FX (Bio-Rad). Left facing triangle indicates migration of unextended dT20 primer and asterisk indicates bromophenol blue dye front. C. RT MS2-specific primer extension. 5′-labeled primer was annealed to MS2 RNA and incubated 10 minutes at 37°C and then 30 minutes at 65°C in the presence of indicated Pol enzymes with dNTPS (N = A,C,G,T) in manufacturer recommended buffers. Primer extension products were resolved by 5% denaturing PAGE. Lane 1 No <t>RNA+MMLV</t> RT; Lane 2: MS2 RNA No RT; Lane 3 MS2 RNA+MMLV RT, Lane 3 MS2 <t>RNA+3173</t> Pol. Molecular weight in bases indicted. Red Arrow: ∼650 base MMLV extension product. Blue Arrow: ∼715 base PyroScript extension product. Green arrow: Non-templated MMLV reaction product.