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Technical Report #1 Ovation™ RNA Amplification System A Comparison of Gene Expression Levels in Non-Amplified and Amplified RNA BACKGROUND Real-time quantitative PCR (QPCR) to examine the accuracy, reproducibility from Integrated DNA Technologies (IDT) and sensitivity of the Ribo-SPIA according to Primer Express® 2.0 amplification process used in the (Applied Biosystems) rules or purchased ™ Ovation RNA Amplification System. from Assays-on-Demand™ (Applied Using real-time PCR, we examined Biosystems). Contact NuGEN Technical these parameters by measuring Services ([email protected]) for expression levels of a panel of genes information regarding primer and probe in both amplified and non-amplified sequences and experimental design. RNA preparations. technology measures the relative expression levels of specific genes RESULTS AND CONCLUSIONS MATERIALS AND METHODS across many samples. Recommendations Using 20 ng starting total RNA, the for each QPCR reaction include a Total RNA samples used in this experi- Ovation™ RNA Amplification System minimum of 10,000 template copies or ment were Universal Human Reference yielded an average of 2.0 µg of cDNA 1-100 ng of total RNA (Applied Biosystems RNA (UHR, Stratagene cat. #74000), a product with a standard deviation of 0.1 Protocol). Thus, to study a series of RNA mixture from 10 cancer cell lines, µg. This yield was within the expected gene transcripts in replicates, microgram and human skeletal muscle (SKMU, range of 1.5 to 2.0 µg of cDNA from 5 quantities of total RNA may be required. BD Clontech cat. #636534). to 100 ng of starting total RNA. limited and this restricts the number of Triplicate reactions of each sample type An assessment was made of the fidelity gene transcripts that can be studied. were used in both amplified and non- of amplification of differentially expressed Compounding the matter, analyses of amplified reactions. Twenty ng of starting genes. QPCR Ct values were measured low abundance transcripts require large total RNA were amplified with the for 39 gene transcripts in both amplified amounts of total RNA input for each Ovation™ RNA Amplification System and non-amplified UHR and SKMU RNA. QPCR measurement. (NuGEN, cat. #2200-48). For the non- The Ct value reflected the expression of Often the size of the starting sample is amplified cDNA product, 4 µg of total a specific transcript in a given RNA One approach to enhancing the sensitivity RNA was reverse transcribed using preparation. The difference in expression of QPCR analyses is pre-amplification of RETROscript™ (Ambion, cat. #1710). of a gene between RNA preparations total RNA; enriching the amount of mRNA Both cDNA products subsequently were (UHR vs. SKMU) was determined by species while successfully maintaining diluted 1:10 in 1 x TE prior to QPCR. subtracting the Ct value in UHR RNA relative gene expression levels compared Eight ng of amplified cDNA and 20 ng from the Ct value in SKMU RNA. This to the starting material. Additionally, of RNA equivalent (non-amplified) was difference in Ct values (∆Ct) reflects the pre-amplifying total RNA addresses added to each QPCR reaction. Real-time differential expression of a gene between sample limitations, creating opportunities PCR was performed on a panel of 39 the two RNA preparations. Delta Ct values for researchers to answer questions genes. Genes were chosen for varying (and hence differential expression) were previously limited by sample size. levels of gene expression and primer calculated for all 39 gene transcripts in positions at varying distance from the amplified and non-amplified RNA. A Up to 10,000 fold amplification of mRNA poly A tail. Data was analyzed according strong correlation of resultant ∆Ct values species in a total RNA preparation has been to Applied Biosystems User Bulletin #2. (which are a measurement of differential described for RiboSPIA™ amplification gene expression) was obtained with QPCR primers and probes (Dual amplified and non-amplified RNA results from a series of studies designed Labeled DNA probes) were synthesized (Figure 1), with a linear regression DISCOVERY technology. This report describes the imagine more from less NuGEN Technologies, Inc. Toll Free 888.654.6544 www.nugeninc.com Technical Report #1 Ovation™ RNA Amplification System correlation coefficient (R2) of 0.97. and two QPCR assays per sample. The that the QPCR primer/ probe sets These results demonstrate that differen- Ct results for the panel of 39 genes are (amplicons) were from the 3’, poly A tail. tial expression of genes (spanning low shown in Figure 3. The mean Cts were Similar Ct values were obtained between to high abundance levels in both UHR plotted with corresponding standard amplified and non-amplified RNA, and SKMU RNA, as measured by real- deviations (Figure 3). (Note that the regardless of amplicon location. These time PCR,) was maintained after standard deviations were determined data demonstrate that with amplified amplification with the Ovation RNA and plotted for all gene transcripts; cDNA product, QPCR primer and probe Amplification System however, due to the high level of repro- sets can be chosen up to at least ducibility, many of the standard deviation 1.5 kb from the 3’ poly A tail. A comparison of expression levels in determinations were very small and amplified and non-amplified human subsequently difficult to visualize in Maintenance of relative gene abundance skeletal RNA was made. Individual Ct Figure 3). The data shows that high, levels across a broad spectrum of genes values were determined for each of the medium and low expressing transcripts is essential in any gene expression 39 genes in amplified and non-amplified can be confidently amplified with an methodology requiring amplification of RNA. The resulting Ct values were plot- average, coefficient of variation (CV) mRNA. The Ovation™ RNA Amplification ted and demonstrated a high level of of 1.9%, for the 39 genes. System maintains relative gene correlation with a R2 value of 0.90 expression assuring that expression data (Figure 2). Regression analysis of Ct QPCR amplicons can be designed at following amplification is representative values for amplified and non-amplified various locations on a gene transcript. of the gene expression in the starting RNA demonstrated a linear amplification In the current study, amplicon locations material. Furthermore, pre-amplification profile with a slope of 0.97. These results for the 39 genes were chosen up to enhances the abundance of all mRNA demonstrate linear amplification over 1.5 kb from the 3’ poly A tail. Figure 4 species, which results in an increased a broad dynamic range. depicts the Ct values for a representative sensitivity with subsequent QPCR set of the gene transcripts in amplified analyses. This increase in sensitivity Reproducibility of amplification was and non-amplified samples. The Ct values reflects the enrichment of mRNA species determined using triplicate RNA samples are plotted as a function of the distance in the sample after amplification. 15 Figure 1. Linear correlation of differential gene expression in non-amplified and amplified RNA Non-Amplified cDNA (∆Ct UHR - SKMU) R2 = 0.97 10 Differences in differential gene expression between 5 UHR and human skeletal muscle RNA are presented as ∆Cts. For non-amplified product, 4 µg of total 0 -25 -20 -15 -10 -5 RNA was reverse transcribed prior to QPCR. 0 -5 5 10 15 20 Approximately 20 ng of reverse transcribed RNA equivalents was loaded into each PCR well. For amplified product, 20 ng of total RNA was used to -10 produce SPIA™ amplified cDNA. The product was diluted 1:10 to 4 ng/µl of cDNA product and 2 µl -15 (8 ng) was loaded into each QPCR reaction. The ∆Ct values for amplified RNA and non-amplified RNA -20 Amplified cDNA (∆Ct UHR - SKMU) were plotted on the x-and y-axis, respectively. A high level of correlation (R2 = 0.97) was obtained. 2 Technical Report #1 Ovation™ RNA Amplification System 50 Figure 2: Correlation of Ct values between amplified and non-amplified skeletal muscle RNA 2 R = 0.90 Non-Amplified cDNA Ct 40 Ct values were determined for a panel of genes in Ribo-SPIA™ amplified and non-amplified skeletal muscle RNA. Amplified versus non-amplified values 30 were compared. The location of the linear regression line (black) with a slope of 0.97 indicates linear amplification lowers Ct values for amplified 20 compared to non-amplified material. 10 0 0 10 20 30 40 50 Amplified cDNA Ct 40 Figure 3: Reproducibility of amplification in human skeletal muscle RNA Threshold Cycle (Ct) Mean Ct values and standard deviations of the 39 genes are shown. Each point represents an average of 6 data points obtained from 3 separate 30 amplification reactions, which were followed up with duplicate QPCR reactions. 20 10 0 10 20 30 40 Gene Number Figure 4: Primer and probe sets can be chosen up to 1.5 kb from poly A tail Threshold Cycle (Ct) 40 Locations of QPCR amplicons for amplified and 30 non-amplified cDNA for 12 different gene transcripts are depicted. The x-axis represents the number of bases the amplicon is located from the poly A tail. 20 Ct values were determined for both amplified and non-amplified cDNA. 10 0 0 250 500 750 1000 1250 1500 Number of Bases from the Poly A Tail Amplified Non-Amplified NuGEN Technologies, Inc. • 821 Industrial Road, Unit A • San Carlos CA 94070 USA • Toll-free 888.654.6544 • Tel 650.590.3600 • www.nugeninc.com © Copyright 2004, NuGEN Technologies, Inc. This product and methods using this product are covered by pending patent applications including the following patent publications: WO 02/072772: US2003/0017591 A1. NuGEN™, Ovation™, Ribo-SPIA™, and SPIA™ are trademarks or service marks of NuGEN™ Technologies, Inc. All other marks appearing in these materials are marks of their respective owners. 3