Make Quantitative RT-PCR templates with Full Spectrum Amplification Technology

Measuring the gene expression levels from small RNA samples is a very important technique in answering today’s functional genomics questions. Unfortunately, small sample quantities can limit the number of genes one can analyze by quantitative RT-PCR. The Full Spectrum RNA Amplification Kit from System Biosciences was designed to address this issue. Using proprietary amplification technology, the Full Spectrum method uniformly amplifies RNA from samples as small as 5 nanograms. In a single tube, in under three hours, it is possible to generate enough amplified template for hundreds of qRT-PCR reactions. The unique sequence independent priming method used in the Full Spectrum kit enables the analysis of the entire mRNA transcript, even in degraded mRNA samples. In comparison, standard T7/IVT amplification methods which use oligo-dT priming amplify only the extreme 3’ end of the transcript, making analysis of alternative splicing impossible. Aside from this 3’ end bias, T7/IVT methods take days to perform, require multiple purifications, and require high quality non-degraded RNA samples. .

Full Spectrum RNA Amplification Maintains Relative Transcript Quantities

Your RNA amplification method must amplify all transcripts equally, so you have confidence that you’re measuring relevant biological expression levels. To demonstrate the uniformity of amplification achieved with the Full Spectrum RNA Amplification Kit, expression levels of 50 human genes were measured before and after amplification of 20 ng total RNA, and the Ct values were plotted in Chart 1A. The identical experiment was performed using a T7/IVT method with the exception that 50 ng total RNA was used, and the results were plotted in Chart 1B. As shown, the Full Spectrum method (R = 0.940) produces results that meet and exceed those of the T7/IVT method (R = 0.770). The data supports the assertion that the Full Spectrum method maintains relative transcript quantities during the amplification process.

Chart 1. Plot of Ct values for 50 genes obtained from non-amplified and amplified cDNA with the Full Spectrum method (1A) and T7 IVT method (1B).

Identify Differentially Expressed Genes with Full Spectrum RNA Amplification

To demonstrate the ability of the Full Spectrum method to identify differentially expressed genes by real time qPCR, we compared Ct values obtained from two different tissue sources. Of the 50 total genes we analyzed in Chart 1, TaqMan assays were prepared for 38. The first of these tissue sources was human universal RNA, which is a mixture of several tissues, and the second was kidney. Again, we compared these results with both amplified and non-amplified mRNAs and also compared mRNA amplified by the Full Spectrum method and the T7/IVT method. We then plotted the differences in Ct values by subtracting the Ct value obtained from universal RNA from the obtained kidney Ct (Charts 2A and 2B). As shown, the Full Spectrum RNA Amplification Kit identifies the same differentially expressed genes as the T7/IVT method, but with a much higher correlation coefficient.

Chart 2. Plot of ΔCt values. Kidney RNA Ct – universal RNA Ct from non-amplified and amplified cDNA with Full Spectrum (2A) and T7 IVT (2B) methods. Color coded dots represent the same gene on both plots.