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Ovation® Whole Blood Solution

Fast and simple method for generating amplified cDNA from 5–100 ng of whole blood total RNA in about 4 hours.
Overview
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Tab 01 / Overview
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The Ovation® Whole Blood Solution is comprised of a set of optimized kits and protocols to enable gene expression analysis of whole blood total RNA without the need for globin reduction procedures.

The Ovation® Whole Blood Solution utilizes two kits for amplification of whole blood total RNA and one kit for the preparation of labeled target. The amplification process is powered by SPIA®, a proprietary technology developed by Tecan.

 

Selection guide
  • Ovation® RNA Amplification System V2 provides a fast and simple method for generating amplified cDNA from 5–100 ng of whole blood total RNA in about 4 hours.
  • Ovation® WB Reagent is a companion reagent for use with the Ovation RNA Amplification System V2. The incorporation of this reagent into the procedure significantly increases amplified cDNA yields.
  • Encore® Biotin Module is used for fragmentation and labeling of amplified cDNA in 2 hours for analysis on Affymetrix GeneChip® arrays.
  • Single-day workflow enables analysis on Affymetrix GeneChip® arrays or amplified cDNA is also suitable for qPCR analysis or sample archiving.

The Ovation® Whole Blood Solution kits provide optimized reagent mixes and protocols to process 60 RNA samples and are also available in 96 reaction sizes for automation workflows.

Applications

  • Gene expression
  • qPCR
  • Archiving




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Tab 02 / Highlights
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High reproducibility with Affymetrix arrays

  • Excellent performance metrics with respect to scaling factors, % Present calls, background and acceptable 3’/5’ ratios

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.

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 Array

Amplification Type

Amplification Type

Initiated at the 3’ end

Starting Material

Starting Material

Total RNA

Input Amounts

Input Amounts

5 – 100 ng




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Tab 04 / Faq
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FAQ

Getting started

Has Tecan performed reproducibility studies on the Encore Biotin Module?

Yes. Our studies have included sample-to-sample, lot-to-lot and operator-to-operator reproducibility. Refer to the Tecan Technical Report on Encore Biotin Module Performance for a summary of our performance data.

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 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.

Do I need to use high quality total RNA?

Yes. Use of lower quality RNA may result in poor performance. One approach to determining RNA quality is the Agilent Bioanalyzer’s RNA Integrity Number (RIN). Clean RNA with a RIN score greater than 7 should amplify well.

How much total RNA input do I need for amplification?

We recommend RNA input of 5–100ng RNA from whole blood or total cellular RNA; however, due to the high levels of globin present in whole blood RNA samples, we advise using inputs of 20 ng or higher when feasible.

Do you recommend any specific whole blood RNA isolation methods?

We do not specifically require one method of RNA preparation, however, for whole blood samples the QIAGEN PAXgene products for stabilization and RNA isolation have been validated and are compatible with the Ovation Whole Blood Solution. In general, any method that yields high quality, non-degraded RNA that is free of organic solvents and contaminants should work well.

Can the Encore Biotin Module be used for fragmentation and labeling of RNA?

No.

General workflow

Where can I safely stop in the Encore Biotin Module protocol?

We do not recommend stopping at any step of the protocol.

What are the recommended storage conditions for the labeled cDNA?

The labeled cDNA should be stored at –20°C. Ensure the vials are well sealed and avoid multiple freeze/thaw cycles.

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

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

The total yield of cDNA is not directly dependent upon input RNA amount due to upper limit constraints on cDNA production in the reaction.

Should I purify the cDNA before determining the concentration?

Yes. The primers and reagents present in the amplified cDNA will interfere with accurate quantitation. Details on measuring the concentration of cDNA are included in the User Guide.

How do I determine fragmentation success?

You may use an Agilent Bioanalyzer to inspect the size distribution of samples before and after fragmentation as described in Appendix B.

Fragmentation, labeling and hybridization

What materials are provided with the Encore Biotin Module?

The Encore Biotin Module provides all necessary buffers and enzymes for fragmentation and labeling of cDNA generated by the Ovation Whole Blood Solution.

How much fragmented and labeled cDNA does this kit yield?

Since the Encore Biotin Module does not require any purification, the final yield is equal to the amount of cDNA input.

What is the size range of cDNA fragments generated by the Encore Biotin Module?

As measured with an Agilent Bioanalyzer, 80% of product falls below 200 bases with an average peak at 85 bases.

Should I purify the labeled cDNA before hybridization?

No. Purification of the labeled cDNA is not necessary.

Are the array hybridization reagents included in the Encore Biotin Module?

No. Refer to Appendix C for information on recommended array hybridization reagents.

What hybridization and wash protocols do you recommend for Affymetrix GeneChip applications?

Refer to Appendix C for information on array hybridization protocols.




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Tab 05 / Literature
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Literature

Type
Title
No
Technical Report
Ovation Whole Blood Solution
M01050 V2.2
User Guide
M01110 V6.1
Quick Protocol
Ovation Whole Blood Solution
M01155 V3
Safety Data Sheet
 
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For research use only. Not for use in diagnostic procedures.
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