Ovation^® RNA-Seq FFPE Kit

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

The Ovation RNA‑Seq FFPE System provides all necessary buffers, primers and enzymes for first strand synthesis, second strand synthesis and amplification. The kit also provides nuclease-free water and 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, purification columns or SPRI beads are required. Refer to the Appendix in the User Guide for validated purification products and procedures.

Can I use the Ovation RNA-Seq FFPE System for archiving cDNA?

Yes. Amplified cDNA may be stored at –20°C for at least six months.

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

This material can be used with most ds-DNA library preparation kits. For ds-cDNA derived from degraded FFPE RNA, the small fragment size may not be compatible with some transposon-based systems. Please follow the manufacturer’s recommendations for these workflows.

What methods do you recommend for RNA isolation?

We recommend a column-based method for FFPE RNA, including:

• Norgen Biotek Total RNA Purification Kit
• Zymo Research Quick-RNA™ FFPE Kit
• Arcturus^® Paradise^® PLUS FFPE RNA Isolation Kit
• PureLink™ FFPE RNA Isolation Kit
• Qiagen RNeasy FFPE Kit

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 FFPE total RNA do I need for amplification?

We recommend total FFPE RNA input of between 100 and 200 ng. Input amounts outside this range may produce unsatisfactory variable results, especially for highly degraded RNA.

Can I use RNA from sources other than FFPE?

While it is possible to use RNA from other sources with the Ovation RNA-Seq FFPE System, it was developed specifically for use with FFPE total RNA.

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

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

Do you recommend DNase treatment of my total RNA sample?

Yes. When using purified total RNA from FFPE samples, genomic DNA may be amplified as well. For this reason we recommend DNase treatment during RNA purification. Refer to section III.A.4 for more information about DNase treatment of RNA samples.

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

NuGEN's Ovation FFPE RNA-Seq System 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?

You may stop immediately following the SPIA Amplification or after Post-SPIA Modification II prior to final cleanup at the points specifically noted in the protocol. Store reaction products at –20°C.

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 much cDNA can I expect from a single reaction?

You should expect a sufficient yield of cDNA for NGS library preparation.

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

Yes. Generally, higher RNA inputs will result in somewhat higher amplification yields; however, at FFPE RNA inputs above 200 ng the yields may become variable.

What size cDNA is generated by the Ovation RNA-Seq FFPE System?

The amplified cDNA size distribution is highly dependent on the input RNA integrity. Figure 4 in the User Guide shows a typical SPIA cDNA fragment size distribution for intact and FFPE total RNA samples.

Does the Ovation FFPE RNA‑Seq System 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.

Where in my target sequence can I design my qPCR primers?

The Ovation RNA-Seq FFPE System includes random priming and, therefore, qPCR primers can be designed at any location within the mRNA. In order to avoid qPCR interference from possible genomic DNA contamination, we recommend treating your RNA with DNase and designing your amplicons to span an intron. We strongly recommend designing your assays for multiple locations across the transcript with the smallest amplicon size possible, since the starting FFPE RNA is likely to be highly degraded.

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

No. It is recommended that qPCR be performed using the amplified SPIA cDNA prior to the Post-SPIA Modification steps. A small aliquot can be removed immediately following SPIA where noted in the protocol. Refer to Appendix B of the User Guide for recommendations on achieving optimal results.