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Ovation® Pico WTA System V2

Fast and simple method for preparing amplified cDNA from total RNA for gene expression analysis.
Overview
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Tab 01 / Overview
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The Ovation® Pico WTA System V2 is a fast and simple method for preparing amplified cDNA from total RNA for gene expression analysis. Amplification is initiated at the 3’ end as well as randomly throughout the whole transcriptome in the sample, making the system ideal for amplification of degraded RNA. The Ovation® Pico WTA System V2 is powered by SPIA® technology, a rapid and sensitive RNA amplification process developed by Tecan.

Ovation® Pico WTA System V2 provides optimized reagents and a protocol to process 12, 60 or 96 RNA samples.

 

Selection guide

Enable workflow flexibility

  • Robust amplification provides access to a wide range of challenging samples.
  • Whole transcriptome approach enables flexibility in qPCR assay design.

Applications

  • Microarray gene expression analysis
  • qPCR
  • Archiving




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Tab 02 / Highlights
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Analyze the most challenging samples on any microarray platform

  • Single tube protocol completed in less than
  • 5 hours using 500 pg – 50 ng of total RNA
  • Amplification yields 6 – 10 μg of cDNA
  • Amplified cDNA can be fragmented and labeled in less than 2 hours using the Encore® Biotin

Ovation Whole Blood Signal Correlation

Duplicate samples were prepared from 20 ng of human whole blood RNA using the Ovation Whole Blood Solution, according to the product user guides. RNA samples were isolated using the Agencourt RNAdvance™ Blood kit on the Beckman ArrayPlex. Targets were hybridized to Affymetrix GeneChip®U133_plus_2.0 arrays. Results of linear regression analysis show a strong signal correlation of R = 0.992, with 58% present calls, demonstrating very high reproducibility across transcript abundance with only 20 ng of input total whole blood RNA.




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Tab 03 / Specifications
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Specifications

Specification

Specification/Description

Compatible Platform

Compatible Platform

Affymetrix GeneChip Arrays, Illumina BeadChips, Agilent Dual Mode Arrays

Amplification Type

Amplification Type

Whole transcriptome

Starting Material

Starting Material

Total RNA

Input Amounts

Input Amounts

500 pg – 50 ng




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Tab 04 / Faq
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FAQ

Getting started

What materials are provided with the Ovation Pico WTA System V2?

The Ovation Pico WTA System V2 provides all necessary buffers, primers and enzymes for first strand synthesis, second strand synthesis and amplification. It also provides nuclease-free water and, in the case of the 12- and 60-reaction size kits, Agencourt Beads for double-stranded cDNA purification. Beads for the final purification step and for the automation-fill size kit must be purchased separately.

Does the Ovation Pico WTA System V2 provide any labeling reagents?

No. The Ovation Pico WTA System V2 is used to generate SPIA cDNA from small amounts of total RNA, which can be used immediately for qPCR analysis or cDNA storage. The SPIA cDNA produced by this kit may be processed further using an Encore labeling module (for analysis on Thermo Fisher Scientific (formerly Affymetrix) GeneChip arrays) or other supported labeling protocols.

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.

Has Tecan performed reproducibility studies on the Ovation Pico WTA System V2?

Yes. Sample-to-sample, lot-to-lot and operator-to-operator reproducibility tests are conducted.

Is the Ovation Pico WTA System V2 3´ biased?

In this system, oligo dT primers are mixed with random primers for the first strand synthesis of cDNA products. This allows the product to be analyzed on 3‘ expression arrays when used with an appropriate Encore labeling module, or other supported labeling protocol. Additionally, the random primers allow the detection of the entire transcripts when used as a pre-qPCR amplification system.

Can I use the Ovation Pico WTA System V2 for archiving cDNA?

Yes. SPIA 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 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 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.

How much total RNA do I need for amplification?

We recommend using between 500 pg and 50 ng total RNA as starting material. Input amounts outside this range may produce unsatisfactory and variable results.

Do I need to use high-quality total RNA?

RNA samples of high molecular weight with little or no evidence of degradation will amplify very well with this product. However, due to the whole transcriptome amplification approach, lower quality RNA samples and transcripts with a compromised poly(A) tail can also be amplified successfully using the Ovation Pico WTA System V2. However, the RNA should have high purity, and be free of contaminants.

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.

Do you recommend DNase treatment of my total RNA sample?

Yes. For an explanation of DNase requirements see section III.A.4 of the User Guide. You may also find recommended procedures for DNase treatment in the Appendix.

Can I use the Ovation Pico WTA System V2 on bacterial RNA samples?

The Ovation Pico WTA System V2 amplification process has been shown to work with some bacterial RNAs. However, the kit has not been optimized for this purpose.

Are there any tissues that will not work with the Ovation Pico WTA System V2?

We have not encountered any specific RNA sources that will not work with the Ovation Pico WTA System V2. The RNA should have high purity and be free of contaminants.

General workflow

Can I perform fewer than 4 reactions at a time?

We recommend a minimum batch size of four reactions. Smaller batch sizes may result in difficulty with pipetting small volumes. Perform at least 4, 10, or 48 reactions at a time for the 12-, 60-, or 96- (A01) reaction kits, respectively, to ensure enough reagents are available for all reactions in the kit.

How many rounds of amplification are performed with the Ovation Pico WTA System V2?

This System has a single round of amplification. It cannot be used for multiple rounds.

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

Yes. The Ovation Pico WTA System V2 will not work properly with other primers. There is no need to order any primers, as DNA/RNA primers provided in the Ovation Pico WTA System V2 are universal.

What purification methods do you recommend?

  1. For the Second Strand cDNA purification step (pre-amplification) we require the use of the Agencourt Beads provided with the 12- and 60-reaction kits. The A01 kit does not include beads.
  2. Several purification options are available for the final SPIA cDNA cleanup step. These are described in Appendix A of this user guide. Selection of the optimum purification option can depend on many factors. Please contact the Tecan NGS Technical Support team at techserv-gn@tecan.com for assistance in selecting the appropriate option for your application. Refer to section II.B. for ordering information.

Where can I safely stop in the protocol?

The SPIA cDNA can be stored at -20 °C prior to performing the purification. We do not recommend stopping at any intermediate stage of the protocol.

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:

  1. 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.
  2. Prior to purchasing, check the manufacturer’s specifications for minimum and maximum volumes that can be effectively treated.
  3. 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?

  1. Allow the SPRI beads to reach room temperature before use; cold beads result in lower yields.
  2. Ensure that the beads are fully resuspended in solution before adding to the sample.
  3. 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.
  4. 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 at least 6 μg of cDNA from inputs of 500 pg to 50 ng 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 50 ng, the yields may become variable without increasing.

What size cDNA is generated by the Ovation Pico WTA System V2?

The SPIA cDNA size distribution is somewhat dependent on the input RNA integrity. A representative size distribution is given in Figure 3.

How do I measure my SPIA cDNA product yield?

You may use a Nanodrop, Qubit, or standard spectrophotometer. Refer to Section IV, Protocol I in the User Guide for specific guidance.

Fragmentation, labeling and hybridization

How much of the SPIA cDNA from the Ovation Pico WTA System V2 should I use for array labeling?

Please refer to guidelines in the user guide for the Encore Biotin Module.

Using amplified cDNA for qPCR

How many qPCR reactions will I get from one Ovation Pico WTA System V2 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 you will need to use more cDNA per reaction. The user will need to determine how much cDNA to use per reaction depending on the abundance of the gene being interrogated. Note that we recommend purification of the SPIA cDNA prior to qPCR analysis.

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.

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

The Ovation Pico WTA System V2 does not have a 3’ bias and, therefore, 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 input RNA with DNase and designing your amplicons to span an intron.




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Tab 05 / Literature
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Literature

Type
Title
No
Technical Report
qPCR Assessment of Effective RNA Quantity
M01028 V2.1
User Guide
M01224 V6
Quick Protocol
Ovation Pico WTA System V2
M01365 V2
Safety Data Sheet
 

For research use only. Not for use in diagnostic procedures.
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