Ovation® FFPE WTA System

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The Ovation® FFPE WTA System provides a fast and simple method for preparing amplified cDNA from FFPE-derived total RNA.

Amplification is initiated at the 3´ end as well as randomly throughout the whole transcriptome in the sample, making this system ideal for amplification of the severely degraded and chemically modified RNA typically obtained from FFPE samples.

The Ovation® FFPE WTA System is powered by SPIA® technology, a novel and sensitive RNA amplification process developed by Tecan.

Selection guide

Tecan has been a valued partner in the development and commercialization of Afirma® Thyroid FNA Analysis. Tecan’s sample preparation technology is a critical component in addressing the challenges of extracting genomic information from small, fine needle aspirate biopsies. Their dependable supply, quality and excellent customer service provides us with the assurance we need in pioneering the development of novel molecular diagnostic tests.

Bonnie Anderson, Cofounder and CEO Veracyte®

Applications

Gene expression
qPCR
Archiving

Simplify your workflow

Add and incubate workflow
Robust amplification achieved across a range of sample ages

Graph above shows data from Forty-one FFPE-derived ovarian tumor samples. A minimum of 5.8 μg is required for analysis on GeneChip arrays (shown by the red bar) a criteria met by all amplifications in this study.

Generate quality data

Integrity of biological data is maintained
No additional ribosomal reduction step required minimizing the introduction of bias

Targets were prepared from RNA extracted from formalin-fixed, paraffin-embedded tissue (FFPE) from one donor (red) and fresh frozen tissue from a second donor (blue). Each sample was amplified in quadruplicate and hybridized to Affymetrix HG-U133A 2.0 GeneChip arrays. PCA was performed using Partek Genomics Suite software. The green and blue ellipses (NAT and Tumor, respectively) define the boundary of 2 standard deviations from the centroid of each cluster indicating a statistically significant separation of samples based on the disease state of the tissue. This demonstrates that the amplification system maintains the integrity of the biological data.

Specifications

Specification	Specification/Description
Compatible Platform	Compatible Platform Affymetrix GeneChip Arrays, Illumina BeadChips, Agilent Dual Mode Array
Amplification Type	Amplification Type Whole transcriptome
Starting Material	Starting Material Total RNA derived from FFPE tissue
Input Amounts	Input Amounts 50 – 100 ng

FAQ

Getting started

What materials are provided with the Ovation FFPE WTA System?

The Ovation FFPE WTA 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 double-stranded cDNA purification. Beads are not provided for the final purification step.

Does the Ovation FFPE WTA System provide any labeling reagents?

No. This system is used to generate SPIA cDNA from FFPE RNA samples. The SPIA cDNA may be processed further using a Tecan Encore labeling module or other supported labeling protocol.

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 Fragment Analyzer may also be useful for optional analytical tests.

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.

Is the Ovation FFPE WTA System 3´ biased?

In this system, oligo dT primers are mixed with random primers for the first strand synthesis of cDNA products. This allows amplification coverage of the whole transcript. We have tested the system with both degraded and intact RNA on 3´ microarray designs as well as arrays interrogating sequence in all regions of the transcript (i.e., Thermo Fisher Scientific (formerly Affymetrix) GeneChip arrays) with successful results.

Can I use the Ovation FFPE WTA System for archiving cDNA?

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

Input recommendations

What methods do you recommend for RNA isolation?

We recommend a column-based method, including:

Norgen Biotek Total RNA, Soil RNA, or Stool RNA Purification Kits
Zymo Research QuickRNA™ Kit, ZR Fungal/Bacterial RNA MicroPrep™, or ZR Soil/Fecal RNA MicroPrep™
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 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.

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 Tecan NGS Technical Support for more information.

Do I need to use high-quality total RNA?

No. The Ovation FFPE WTA System employs a whole transcriptome amplification approach and was designed specifically for use with highly degraded, lower quality RNA samples.

Can I use RNA from sources other than FFPE?

Yes. While the Ovation FFPE WTA System was developed specifically for use with total RNA from FFPE samples, it is possible to use intact, non-FFPE RNA samples as well. Performance with intact total RNA samples below 2 ng may vary.

How much FFPE derived total RNA do I need for amplification?

We recommend total FFPE RNA inputs in the range of 50 to 100 ng. Input amounts outside this range may produce unsatisfactory variable results, especially for more degraded RNA.

Can DNA be used as input for the Ovation FFPE WTA System?

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

Can contaminating genomic DNA interfere with the amplification performance?

Yes. This system is designed to amplify RNA, but contaminating genomic DNA may amplify during the process. For this reason we recommend DNase treatment during RNA purification.

Can I use the Ovation FFPE WTA System on bacterial RNA samples?

The Ribo-SPIA amplification process may work with some bacterial RNAs. However, the kit has not been optimized or validated for this purpose.

General workflow

Do I need to order specific primers for the amplification?

No. The DNA/RNA primers provided in the Ovation FFPE WTA System are universal.

What purification methods do you recommend?

For the Second Strand cDNA purification step (pre-amplification) we require the use of the Agencourt Beads provided with the kit.
Several purification options are available for the final SPIA cDNA cleanup step. These are described in Appendix A of the user guide. Selection of the optimum purification option can depend on many factors. Please contact the Tecan NGS Technical Support team for assistance in selecting the appropriate option for your application. Refer to section II.B. for ordering information.

SPRI bead purifications

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 Tecan 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.

cDNA quantification and qualification

How much cDNA can I expect from a single reaction?

You should expect 4 to 7 μg of cDNA from 50 to 100 ng total FFPE RNA starting material, if it is of sufficient quality. Yields may be higher using intact, non-FFPE RNA samples. Although yield is an important sample quality indicator, success of a given FFPE sample set in array analysis is best predicted using the RNA Sample Quality Assessment Tool described in the WT-Ovation FFPE System RNA Sample Quality Assessment technical report.

What size cDNA is generated by the Ovation FFPE WTA System?

The amplified cDNA size distribution can vary based on the input RNA integrity. In a whole transcriptome amplification strategy, however, the size of the resulting cDNA is not of significant consequence for use on arrays.

Using amplified cDNA for qPCR

Do you recommend purification of the cDNA prior to qPCR analysis?

Yes. Although this is not absolutely necessary, it is important to be able to quantify the SPIA cDNA. This allows assessment of amplification success based on the amplification yields. It also allows mass normalization of the cDNA into qPCR.

How many qPCR reactions will I get from one Ovation FFPE WTA System amplification?

The number of qPCR reactions depends on the abundance level of the genes being interrogated. For medium- to high-copy genes, the cDNA may be diluted as much as 400-fold, enough for thousands of qPCR reactions. For very-low-copy genes more cDNA must be used per qPCR reaction. We recommend purification of the amplified cDNA prior to qPCR analysis.

Literature

Type

Title

Technical Report

Microarray Performance Comparison of Ovation FFPE WTA System and the Genisphere Sensation RNA Amplification Kit

M01307 V1.2

User Guide

Ovation FFPE WTA System

M01205 V5

Safety Data Sheet