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Rapid and sensitive whole transcriptome RNA amplification process for preparing amplified cDNA from total RNA or poly(A) selected mRNA.
The look and feel of the user guide for Ovation RNA-Seq System V2 (M01206 v9) has been updated. The changes to the user guide are primarily in wording and organization to improve clarity. There are no changes to the protocol.
cDNA synthesis, also known as reverse transcription, generates complementary DNA (cDNA) from an RNA template. Synthesized cDNA is ready to be used for multiple downstream applications including RNA-Seq and archival storage for future analyses.
The Ovation RNA-Seq System V2 provides a rapid and sensitive whole transcriptome RNA amplification process for preparing amplified cDNA from total RNA or poly(A) selected mRNA. This kit provides high-quality cDNA samples from as little as 500 pg to 100 ng of total RNA in just four and a half hours.
This kit features our unique amplification technology (single primer isothermal amplification (SPIA)) which allows the ability to generate more molecules of cDNA compared to conventional reverse transcription methods. For challenging samples, it is important that the sequencing dataset appropriately captures the limited genomic information available in the biological samples. The SPIA cDNA amplification expands this limited information, improving the sensitivity of downstream applications like NGS, in particular for rare transcript detection. The product is particularly useful for the detection of rare transcripts, viruses, and unknown pathogens.
With over 900 publications and applications in multiple areas of life science studies, this technology has been proven to generate cDNA from many challenging samples including COVID-19 samples from nasal swabs.
The cDNA produced by the Ovation® RNA-Seq System V2 may be used as input for Tecan’s Ovation® library systems or used with other library construction kits suitable for double-stranded cDNA samples.
Concordance plots demonstrating reproducibility of the integrated workflow using Ovation® RNA-Seq System V2 with Tecan’s Ovation® Rapid Library System with differing amounts of SPIA cDNA input to the library construction workflow. RPKM plots average R ≥ 0.9 for different library inputs.
Data was generated using 2 ng of Total RNA from either Human Brain (MAQC B) or Universal Human Reference (MAQC A) input to the Ovation® RNA-Seq System V2 followed by library construction using the Encore NGS Library System I. The resulting libraries were sequenced on the Illumina Genome Analyzer IIx with 40 bp single-read sequencing.
Total RNA from Human Brain (MAQC B, 2 ng) was amplified using either the original Ovation® RNA-Seq System or the Ovation® RNA-Seq System V2 and libraries constructed using the Encore® NGS Multiplex System I. Single-read sequencing results were obtained using the Illumina Genome Analyzer IIx platform with 40 bp reads. Results depicted here are for Beta-Actin gene.
Total RNA from Human Brain (MAQC B, 2.ng) was amplified using either the original Ovation® RNA-Seq System or the Ovation® RNA-Seq System V2 and libraries constructed using the Encore® NGS Multiplex System I. Single-read sequencing results were obtained using the Illumina Genome Analyzer IIx platform with 40 bp reads. Results depicted here are for GAPDH gene.
Differences between Log2 transformed expression values for MAQC A and MAQC B samples, RNA-Seq data using 2 ng total RNA as input are plotted on the X axis, qPCR data are plotted on the Y axis. 659 TaqMan probes that uniquely map to the RefSeq annotations used in the RPKM calculations are represented. RPKM stands for Reads Per Kilobase of exon model per Million mapped reads. The RPKM measure of read density reflects the molar concentration of a transcript in the starting sample by normalizing for RNA length and for the total read number in the measurement.
Illumina HiSeq, MiSeq, NextSeq, MiniSeq
500 pg – 100 ng
The Ovation RNA‑Seq System V2 provides all necessary buffers, primers and enzymes for first strand synthesis, second strand synthesis and amplification. The kit also provides nuclease-free water and Beckman Coulter Agencourt Beads for second strand reaction cDNA purification. Beads are not provided for the final purification step.
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. A system for fragmentation, such as a Covaris system, is required for use with most downstream library preparations.
For the SPIA cDNA purification step, we recommend using the QIAGEN MinElute Reaction Cleanup Kit. Refer to Appendix A in the User Guide other purification options.
Yes, Ovation RNA-Seq System V2 generates ds cDNA that can be used for all of these purposes. Resulting cDNA may be stored at -20C for at least six months following purification.
Ovation RNA-Seq System V2 can be used with any ds-DNA library preparation kit.
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.
The Ovation RNA‑Seq System V2 can be used with purified total RNA in the range of 500 pg to 100 ng. Input amounts outside this range may produce unsatisfactory and variable results.
rRNA depletion or Poly(A) enrichment is not required. The input range of 500 pg to 100 ng refers to total RNA.
Yes, if you are primarily interested in sequencing reads from mature coding transcripts then poly(A) RNA may be substituted for total RNA. Tecan has demonstrated that poly(A) RNA can be isolated from 5 to 500 ng of total RNA and used as input into the Ovation RNA-Seq System V2 protocol with good results. We recommend using at least 500 pg of isolated poly(A) RNA.
No. The Ovation RNA-Seq System V2 is designed to amplify RNA, not DNA.
The Ovation RNA-Seq System V2 is designed to work with purified total RNA. When using purified total RNA, samples should be of high molecular weight with little or no evidence of degradation. While it is impossible to guarantee the highest levels of performance when using RNA of lower quality, this system should allow the successful analysis of somewhat degraded samples. With such samples, users may experience lower amplification yields and should plan to use relatively higher RNA inputs.
Yes. When using purified total RNA samples, contaminating genomic DNA may amplify during the process. For this reason we recommend DNase treatment during RNA purification.
The Ovation RNA-Seq System V2 has been optimized for use with mammalian samples. The system should work with most non-mammalian samples, however, you may observe a higher percentage of rRNA than is seen with mammalian samples.
We recommend a minimum batch size of 4 reactions. Smaller batch sizes may result in difficulty pipetting small volumes and lead to poor performance.
A minimum amount of overage should be used in master mixes to ensure the full nominal number of reactions in the kit. The amount of overage needed depends on sample batch size, pipetting accuracy, and viscosity of reagents.
We have found that 10% extra volume in the master mixes in this kit is sufficient for most experiments.
This System performs a single round of amplification and is not designed to support multiple rounds of amplification.
Yes. The Ovation RNA‑Seq System V2 will not work properly with other primers.
Tecan’s Ovation RNA-Seq System V2 does not selectively deplete ribosomal RNA. You may, however, observe a reduction in the amount of ribosomal RNA content with this system when used on mammalian samples.
You may safely stop after the SPIA Amplification step or final SPIA cDNA Purification and store the cDNA at -20 °C.
For the final SPIA cDNA purification step we recommend using the QIAGEN MinElute Reaction Cleanup Kit. Alternative purification methods can be found in Appendix A of the User Guide.
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:
Refer to the Measuring SPIA cDNA Yield and Purity protocol on page 19 in the User Guide for guidance.
You should expect a minimum yield of 2.5 μg of SPIA cDNA from a starting input of 500 pg of good quality total RNA.
Yes, higher RNA inputs will produce higher yields. However, at inputs of above 100 ng, the yields may become variable without increasing.
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.
Yes. Refer to Appendix B of the User Guide for guidelines.
Figure 3 (page 27) in the User Guide shows a representative fragment distribution pattern obtained with 2 ng of high quality Universal Human Reference and human brain total RNA samples.
Yes. qPCR can be performed on the final SPIA cDNA before or after purification. Guidelines for qPCR analysis of SPIA cDNA can be found in Appendix D of the User Guide.
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