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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.




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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.

  • 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

Is the Ovation RNA Amplification System V2 amplification 3’ biased?

Yes. Since the Ovation RNA Amplification System V2 primes the poly(A) tail of transcripts, it is 3’ biased, resulting in coverage up to a range of 1500 bases from the 3’ poly(A) tail.

Has Tecan performed reproducibility studies on the Ovation RNA Amplification System V2?

Yes. Our studies have included sample-to-sample, lot-to-lot, and operator-to-operator reproducibility.

Can I use the Ovation RNA Amplification System V2 for archiving cDNA?

Yes. Resulting cDNA may be stored at –20°C following purification for at least six months. Ensure the vials are well sealed and avoid multiple freeze/ thaw cycles.

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.

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.

Can I use mRNA instead of total RNA as starting material?

Purified poly(A)+ RNA has been successfully used as input to the Ovation RNA Amplification System V2. It may be necessary to reduce the input of mRNA to a level comparable to the mRNA present in 5 ng to 100 ng of total RNA.

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

No. The Ovation RNA Amplification System V2 is designed to use total RNA as input.

Are there any tissues that will not work with the Ovation RNA Amplification System V2?

We have not encountered any good-quality, clean RNA samples containing poly(A)+ RNA that will not work with the Ovation RNA Amplification System V2.

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.

Does the Ovation RNA Amplification System V2 generate product in a no-RNA reaction?

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.

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

The Ovation Amplification System V2 performs a single round of amplification in fewer than 4 hours. Our products are designed to provide high sensitivity through robust amplification without necessitating a second round of amplification.

Do I need to order specific primers for the amplification?

No. The chimeric DNA/RNA primers provided with the Ovation RNA Amplification System V2 kits are universal. There is no need for additional primers.

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

Yes. The Ovation RNA Amplification System V2 was designed and optimized to work with the primers provided. The use of other primers with the Ovation RNA Amplification System V2 is not supported.

Do I need to change the sense of the cDNA for use on oligo arrays?

The Ovation RNA Amplification System V2 generates antisense cDNA. There is no need to change the sense.

Where can I safely stop in the Ovation RNA Amplification System V2 protocol?

You may safely stop after SPIA amplification or after purifying the amplified cDNA and store the cDNA at –20°C. After processing with the Encore Biotin Module, the fragmented and labeled cDNA may also be stored at –20°C for short term storage or at –80°C for longer term storage.

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.

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.

What is the amplification efficiency of the Ovation RNA Amplification System V2?

Based on qPCR results of a collection of housekeeping genes, amplification efficiency ranges from 1,000- to 10,000-fold or higher depending on the input amount.

What is the expected cDNA yield from one reaction of the Ovation RNA Amplification System V2 used with the Ovation WB Reagent?

For a standard reaction the expected yield is 5–12 μg of amplified cDNA.

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.

What is the size range of cDNA generated by the Ovation RNA Amplification System V2?

As measured with an Agilent Bioanalyzer, the majority of amplified SPIA cDNA is 200–2000 bases long. After fragmentation, 80% of the cDNA falls below 200 bases with an average peak at 85 bases.

How can I ensure good yields at the cDNA purification step?

In order to maximize yields, we recommend the following:

  • Do NOT use cold water for the elution step. Use the D1 Nuclease-free Water included in the Ovation RNA Amplification System V2 kit at room temperature.
  • Do NOT spin the columns at an incorrect speed. Strictly adhere to the guidelines in the user guide.
  • Use a fresh dilution of ethanol from a fresh stock for any washing steps.
  • Vortex the eluted cDNA sample prior to measuring the absorbance.

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.

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.

For quantitative real time PCR applications, what is the optimal distance from the 3´ poly(A) tail for design of primer probe sets?

Due to the 3’ oriented amplification mechanism of the Ovation RNA Amplification System V2, we recommend primer/probe sets to be designed within the first 1.5 Kb from the poly(A) tail.




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Tab 05 / Literature
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Literature

Type
Title
No
Technical Report
M01050 V2.2
User Guide
M01110 V6.1
Quick Protocol
M01155 V3
Safety Data Sheet
 
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