To evaluate performance, we compared Stratagenes Brilliant single-step QRT-PCR kit to a another commercially available quantitative RT-PCR one-step system by amplifying and detecting a 150-bp region of human GAPDH mRNA using the Molecular Beacon GAPDH Expression Analysis kit. For this comparison, we amplified three concentrations (100, 1, and 0.01 ng) of human total RNA (Methods); however, because the other commercially available kit did not provide a passive reference dye, none was included in this experiment. We found that for each concentration of target RNA, the Ct was comparable or better for the Brilliant kit (data not shown). We assessed the sensitivity of the Brilliant two-step QRT-PCR kit to detect a low-abundance target by amplifying and detecting hypoxanthine phosphoribosyl transferase (HPRT) mRNA from human brain total RNA. For the real-time analysis of the RT-PCR amplification, we used the primers and linear hydrolysis probe from the TaqMan pre-developed assay reagents (PE Biosystems) in the Brilliant Plus two-step QRT-PCR kit. We included UNG and the passive reference dye.The following Ct values were obtained: 22.5 (100 ng), 26.4 (10 ng), 29.1 (1 ng), 32.7 (100 pg), and 37.7 (10 pg) (data not shown). The results show that with the Brilliant Plus two-step quantitative RT-PCR kit, a low-abundance mRNA can be detected from as little as 10 pg of total RNA.
The Brilliant QPCR and QRT-PCR core reagent kits provide highly sensitive and
versatile quantitative assessments for real-time gene quantification and gene
expression analysis. The kits are compatible with linear hydrolysis (TaqMan
probes), molecular beacon, single dye primers, and Scorpions fluorescent
technologies. With the Brilliant QPCR core reagent kit, we detected as few as 10
copies of mouse muscle nicotinic ace