Ovation^® RNA-Seq System V2

What materials are provided with the Ovation RNA‑Seq System V2?

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.

What equipment is required or will be useful?

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 Tapestation 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.

What additional consumables does the user need?

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.

Can Ovation RNA-Seq System V2 be used for qPCR, archiving and NGS?

Yes, Ovation RNA-Seq System V2 generates ds cDNA that can be used for all of these purposes.

What library preparation kits are compatible with the ds-cDNA generated by RNA-Seq System V2?

Ovation RNA-Seq System V2 can be used with any ds-DNA library preparation kit.

What methods do you recommend for RNA isolation?

We recommend a column-based method, including:

• Norgen Biotek Total RNA Purification Kit
• Zymo Research Quick-RNA™ Kits
• Arcturus PicoPure^® RNA Isolation Kit
• Ambion PureLink^® RNA Mini Kit
• Qiagen RNeasy Kits Organic methods such as TRIzol^® Reagent should be subsequently followed with a column-based clean-up method.

Can I use TRIzol^® or other phenol-chloroform based extractions for RNA isolation?

We do not recommend the use of TRIzol^® or similar methods as any carry over 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.

Can I use carrier RNA during RNA isolation?

We do not recommend the use of carriers during RNA isolation. If a carrier is required, please contact Technical Support for more information.

How much total RNA do I need for amplification?

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.

Do I need to perform an rRNA depletion or poly(A) enrichment step before processing samples with the Ovation^® RNA‑Seq System V2?

rRNA depletion or Poly(A) enrichment is not required. The input range of 500 pg to 100 ng refers to total RNA.

Can I use poly(A)+ RNA as an alternative 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. NuGEN has demonstrated that poly(A)+ RNA can be isolated from 5 to 500 ng of total RNA using MPG mRNA Purification Kit (PureBiotech product number MRRK 1010) and input to the Ovation RNA-Seq System V2 protocol with good results. We recommend not using less than 500 pg of isolated poly(A)+ RNA.

Can DNA be used as input for the Ovation RNA-Seq System V2?

No. The Ovation RNA-Seq System V2 is designed to amplify RNA, not DNA.

Do I need to use high-quality total RNA?

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.

Do you recommend DNase treatment of purified total RNA samples?

Yes. When using purified total RNA samples, large amounts of contaminating genomic DNA may amplify during the process. For this reason we recommend DNase treatment during RNA purification.

With RNA from which organisms can the Ovation RNA-Seq System V2 be used?

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.

Can I perform fewer than 4 reactions at a time?

We recommend a minimum batch size of 4 reactions. Smaller batch sizes may result in difficulty pipetting small volumes and lead to poor performance.

How many rounds of amplification are performed with the Ovation RNA‑Seq System V2?

This System performs a single round of amplification and is not designed to support multiple rounds of amplification.

Do I have to use the DNA/RNA primers supplied with the kit?

Yes. The Ovation RNA‑Seq System V2 will not work properly with other primers.

Does the Ovation RNA-Seq V2 System deplete ribosomal RNA?

NuGEN’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.

Where can I safely stop in the protocol?

It is safe to stop after the SPIA Amplification protocol prior to final cleanup at the point specifically noted in the protocol. Store reaction products at –20°C.

What purification methods do you recommend?

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.

What is the difference between RNAClean XP and AMPure XP SPRI beads? Can both be used interchangeably?

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.

What magnetic separation devices do you recommend for the SPRI bead purifications?

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:

• Use a magnet designed for 0.2 mL tubes (PCR tubes), tube strips, or plates. Compared to magnets that are designed for 1.5 mL tubes, these minimize loss that can occur when samples are transferred from one tube to another.
• Prior to purchasing, check the manufacturer’s specifications for minimum and maximum volumes that can be effectively treated.
• Test the magnet with a mock purification to ensure the magnet will effectively clear the solution under the conditions in the NuGEN workflow. This is also helpful to gain familiarity with the purification workflow.

How can I ensure maximum recovery of sample from the SPRI bead purification?

• Allow the SPRI beads to reach room temperature before use; cold beads result in lower yields.
• Ensure that the beads are fully resuspended in solution before adding to the sample.
• Always use fresh ethanol during the washing steps. When preparing the ethanol, measure out the ethanol and water separately to ensure the desired ethanol concentration is obtained.
• Mix the bead suspension and sample thoroughly to ensure maximum binding of the samples to the beads.

How do I measure my SPIA cDNA yield?

Refer to the Measuring SPIA cDNA Yield and Purity protocol on page 19 in the User Guide for guidance.

How much SPIA cDNA can I expect from a single reaction?

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.

Is the cDNA yield dependent upon the quantity of total RNA input?

Yes, higher RNA inputs will produce higher yields. However, at inputs of above 100 ng, the yields may become variable without increasing.

Does the Ovation RNA‑Seq System V2 generate product in the absence of RNA input?

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.

Can I use an Agilent Bioanalyzer or Tapestation to evaluate the amplification products?

Yes. Refer to Appendix B of the User Guide for guidelines.

What size cDNA is generated by the Ovation RNA‑Seq System V2?

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.

Can I use the final purified SPIA cDNA for qPCR analysis?

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.