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Fast, simple and sensitive method for preparing amplified cDNA from total RNA for gene expression analysis.
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Tab 01 / Overview
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The Ovation® RNA Amplification System V2 provides a fast, simple and sensitive method for preparing amplified cDNA from total RNA for gene expression analysis. Amplification is initiated at the 3’ end of the transcript and yields microgram quantities of amplified cDNA. The Ovation® RNA Amplification System V2 is powered by SPIA® technology, a proprietary amplification process developed by Tecan.
The Ovation® Whole Blood Solution utilizes two kits for amplification of whole blood total RNA and one kit for the preparation of labeled target. The amplification process is powered by SPIA®, a proprietary technology developed by Tecan.
The Ovation® RNA Amplification System V2 provides optimized reagent mixes and a protocol to process 12 or 60 RNA samples and is also available in a 96 reaction size for automation workflows.
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Tab 02 / Highlights
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5 ng of HeLa RNA, amplified, fragmented and labeled in duplicate, as previously described show high signal correlation of R2 of 0.994 and cell concordance of 93.2% demonstrating very high level of reproducibility between independently processed samples.
5 ng of HeLa RNA, amplified, fragmented and labeled in duplicate, and hybridized to GeneChip HG-U133A 2.0 Arrays, show robust and reproducible array metrics.
Using a tissue mixing model, amplification of 5 ng of 100% Placental RNA sample vs a 50% mix of Placental and Spleen RNA and consequent qPCR assays of a panel of 18 Placenta-specific genes showed that the expected 2-fold change is observed and maintained across the different transcripts, the right-most 4 genes in the graph are house-keeping genes, and the very right-most gene, RPL35-1 was used as a normalization gene.
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Graph above shows Amplified cDNA using 50, 20, and 5 ng of total HeLa RNA show consistent profile and size distribution across RNA input range upon analysis of Agilent Bioanalyzer traces.
50, 20 and 5ng of HeLa total RNA were amplified in triplicate. qPCR results for 7 transcripts show reproducible and consistent results with and without purification of amplified cDNA. Yields, show on the left axis of the graph also show highly reproductible degrees of amplification across inputs.
5ng of UHR total RNA was amplified in triplicate for 3 different lots of the Ovation RNA Amplification System V2. qPCR results for 5 genes show reproducible and consistent amplification. Amplified cDNA yields, shown on the left axis of the graph, also show consistent amplification across multiple lots.
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Tab 03 / Specifications
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Specification |
Specification/Description |
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Compatible Platform |
Compatible Platform Affymetrix GeneChip Arrays, Illumina BeadChips, Agilent Dual Mode Array |
Amplification Type |
Amplification Type Initiated at the 3’ end |
Starting Material |
Starting Material Total RNA |
Input Amounts |
Input Amounts 5 – 100 ng |
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Tab 04 / Faq
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The Ovation RNA Amplification System V2 provides all necessary buffers, primers and enzymes for first strand synthesis, second strand synthesis and SPIA amplification. The kit also provides nuclease-free water.
Required equipment includes a microcentrifuge, pipettes, vortexer, a thermal cycler, a spectrophotometer and a magnetic plate for 0.2 mL tubes, strips, or plates. An Agilent Bioanalyzer or Fragment Analyzer may also be useful for optional analytical tests.
For the SPIA cDNA purification step, purification columns or SPRI beads are required. Please refer to page 16 of the User Guide for purification options, as well as page 5 for other consumables.
Yes. Since the Ovation RNA Amplification System V2 primes the poly(A) tail of transcripts, it is 3’ biased, resulting in coverage up to a range of 1500 bases from the 3’ poly(A) tail.
Yes. Our studies have included sample-to-sample, lot-to-lot, and operator-to-operator reproducibility.
Yes. Resulting cDNA may be stored at –20 °C following purification for at least six months. Ensure the vials are well sealed and avoid multiple freeze/ thaw cycles.
We recommend a column-based method, including:
Organic methods such as TRIzol® Reagent should be subsequently followed with a column-based clean-up method.
We do not recommend the use of TRIzol or similar methods as any carryover of organic solvents may inhibit downstream enzyme activity. If using TRIzol extracted RNA, we recommend using a column-based purification of the RNA prior to input into the kit.
We do not recommend the use of carriers during RNA isolation. If a carrier is required, please contact Tecan NGS Technical Support for more information.
Yes. Use of lower quality RNA may result in poor performance. One approach to determining RNA quality is the Agilent Bioanalyzer’s RNA Integrity Number (RIN). Clean RNA with a RIN score greater than 7 should amplify well.
We recommend staying within the specified range of 5 to 100 ng of total RNA starting material. We often suggest 20 ng input as an appropriate starting point. Input greater than 100 ng may adversely affect amplification.
The Ovation RNA Amplification System V2 has been validated for total RNA input amounts of 5 to 100 ng. Using input quantities outside the recommended range will affect cDNA yields.
It is important that RNA input amounts be kept within the 5 to 100 ng range recommended; therefore, we do not recommend omitting quantification of input RNA.
Purified poly(A) RNA has been successfully used as input to the Ovation RNA Amplification System V2. It may be necessary to reduce the input of mRNA to a level comparable to the mRNA present in 5 ng to 100 ng of total RNA.
The Ovation RNA Amplification System V2 is designed to amplify mRNA, not DNA. Please contact Tecan NGS Technical Support for more information on our SPIA-based DNA amplification product offerings.
The Ovation RNA Amplification System V2 relies on the presence of a poly(A) tail for priming. Therefore, it will not amplify most prokaryotic RNA.
We have not encountered any good-quality, clean RNA samples containing poly(A) RNA that will not work with the Ovation RNA Amplification System V2.
Due to the high sensitivity inherent in our amplification systems a non-specific product can be generated in the absence of input RNA. However, in the presence of even a very small amount of RNA the amplified cDNA has been demonstrated to be specific. To minimize the potential for carryover of non-specific amplification products into future reactions, we recommend using a low-input (i.e. 50-100 pg) rather than a no-input (i.e. negative) control.
The Ovation RNA Amplification System V2 performs a single round of amplification in fewer than 4 hours. Our products are designed to provide high sensitivity through robust amplification without necessitating a second round of amplification.
No. The chimeric DNA/RNA primers provided with the Ovation RNA Amplification System V2 kits are universal. There is no need for additional primers.
Yes. The Ovation RNA Amplification System V2 was designed and optimized to work with the primers provided. The use of other primers with the Ovation RNA Amplification System V2 is not supported.
cDNA purification is required for microarray applications. cDNA for use in with an Encore labeling module or other supported microarray labeling protocol must be purified to allow for accurate quantification of cDNA as well as ensure proper performance of the labeling method. If the amplified cDNA is to be used solely for qPCR analysis alone, then purification is optional. Purification, however, will enable mass normalization of cDNA input into the qPCR reaction, potentially reducing variability.
The amplified cDNA may be stored at –20 °C. Ensure the vials are well sealed and avoid multiple freeze thaw cycles.
You may safely stop after the SPIA Amplification step or final SPIA cDNA Purification and store the cDNA at -20 °C.
RNAClean XP beads are certified to be RNase and DNase free. We have tested both RNAClean XP and AMPure XP beads in our kits and observe no difference in performance between products.
Due to the large number of commercially available magnets, we do not have a comprehensive list of compatible products. However, many magnets are compatible. As long as the magnet is strong enough to clear the solution of magnetic beads, it can be applied to the system. We have the following guidelines for selecting a magnetic separation device:
Based on qPCR results of a collection of housekeeping genes, amplification efficiency ranges from 1,000- to 10,000-fold or higher depending on the input amount.
In order to maximize yields, we recommend the following:
For a standard reaction the expected yield is 4–7 μg of amplified cDNA.
The total yield of cDNA is not directly dependent upon input RNA amount due to upper limit constraints on cDNA production in the reaction.
As measured with an Agilent Bioanalyzer, the majority of amplified SPIA cDNA is between 200 bases and 2 Kb in length.
Yes. The primers and reagents present in the amplified cDNA will interfere with accurate quantification. Details on measuring the concentration of cDNA are included in the User Guide.
The cDNA generated with the Ovation RNA Amplification System V2 has been validated on Thermo Fisher (formerly Affymetrix) GeneChip arrays and Agilent Dual-Mode Gene Expression Arrays when used in conjunction with the appropriate Encore labeling module or protocol. Contact Tecan NGS Technical Support for more information.
Due to the 3’ oriented amplification mechanism of the Ovation RNA Amplification System V2, we recommend primer/probe sets to be designed within the first 1.5 Kb from the poly(A) tail.
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Tab 05 / Literature
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Tab 06 / Shop
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Product |
Part Number |
Number of Reactions |
Barcodes |
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Ovation RNA Amplification System V2 |
3100-A01 |
96 |
N/A |
Product |
Part Number |
Number of Reactions |
Barcodes |
Item Price |
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Ovation RNA Amplification System V2 |
3100-A01 |
96 |
N/A |
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For research use only. Not for use in diagnostic procedures.