Ovation™ qPCR System
The Ovation qPCR System enables whole transcriptome gene expression analysis for compromised samples. Ovation qPCR provides a rapid, simple, and sensitive method for preparing in less than four hours micrograms of amplified cDNA from 5 to 50 ng of total RNA with no 3' bias. Amplification is initiated at the 3' end and randomly throughout the whole transcriptome. The amplified cDNA is optimized for the detection of low-, medium-, and high-abundance gene transcripts and is ready for real-time quantitative PCR (qPCR) with no need for purification.
The Ovation qPCR System provides optimized reagent mixes and a protocol to process 24 (cat. # 2210-24) RNA samples.
Detect and quantify more genes with Ovation qPCR System.
cDNA was prepared from Human Jurkat Cell Total RNA (Stratagene, cat.# 540107) for interrogation by quantitative real-time PCR. Unamplified cDNA was prepared in triplicate (RT) directly from 5 µg of total RNA with the SuperScript™ III First-Strand Synthesis System for RT-PCR using random hexamers according to the manufacturer’s instructions (Invitrogen, cat. #18080-051). The Ovation and Ovation qPCR Systems were each used to linearly amplify cDNA in triplicate from 20 ng of total RNA. cDNA equivalents of 100 ng for each method were loaded per sample port as input into the TaqMan® Low Density Immune Profiling Arrays [LDA] (Applied Biosystems, part #4342510). Genes with RT-PCR Cts of 36 or lower were defined as detectable. Twenty percent more genes were detected on the LDA using Ovation qPCR System amplified cDNA than unamplified cDNA. The Ovation system detected the smallest number of genes due to the non-3’-biased design of the 91 TaqMan® Gene Expression Assays present on the LDA, many of which are too distant from the 3’ poly-A tail of the message for the Ovation System cDNA to faithfully represent. These results demonstrate that a significantly higher proportion of genes can be reliably detected and quantified using the Ovation qPCR System than can be detected using standard reverse transcriptase.