l concentration of probe (200 to 400 nM molecular beacon or linear hydrolysis probe); 10 U of StrataScript reverse transcriptase (RT); 2.5 U of SureStart Taq DNA polymerase; and RNA template (total RNA, poly (A)⁺ RNA, or in vitro transcribed RNA). Each experiment was performed with no-RT controls, in which StrataScript RT was omitted from the reaction and with no-template controls. Other commercially available reagents and TaqMan chemistry were tested according to the manufacturers recommendations. The following thermal cycling conditions were used for testing molecular beacons with the Brilliant single-step quantitative RT-PCR core kit: 45C for 30 minutes; 95C for 10 minutes; 40 cycles of 95C for 30 seconds, 55C for 1 minute; followed by 72C for 30 seconds. The following thermal cycling conditions were used for testing linear hybridization probes with the Brilliant single-step quantitative RT-PCR core kit: 45C for 30 minutes; 95C for 10 minutes; 40 cycles of 95C for 30 seconds; followed by 60C for 1 minute. All RT-PCR reactions can be cycled in a temperature cycler designed to detect changes in fluorescence in real time.

Two-step QRT-PCR: All first-strand synthesis reactions were performed in 200-l thin-wall reaction tubes in a 10-l reaction volume with Stratagenes RoboCycler 96 temperature cycler. The RT reaction contained 1X RT buffer, 5.5 mM MgCl₂, 1 mM each dNTP, reverse primer, 100 ng of random oligomers, and RNA template. Reactions in which the RT was omitted were included in each experiment. The reactions were annealed at 25C for 10 minutes [for oligo(dT) or random oligomers], after which 10 U of StrataScript RT was added. The reactions were incubated at 45C for 30 minutes, then at 95C for 5 minutes. An aliquot (1 l) of the first-strand synthesis reaction was added to optical tubes containing 1X Brilliant Plus QPCR core buffer; the optimal concentration of MgCl
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