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Encore™ Biotin Module

Rapid, simple and easily automatable approach for fragmentation and labeling of amplified cDNA
Overview
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Tab 01 / Overview
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The Encore Biotin Module provides a rapid, simple and easily automatable approach for fragmentation and labeling of amplified cDNA generated with Tecan amplification systems. Tecan’s novel and proprietary method combines enzymatic and chemical processes in a two-step fragmentation and labeling process that does not require any purification steps. The resulting labeled cDNA is suitable for hybridization to Affymetrix GeneChip® arrays.

 

Selection guide
  • Fragment and label amplified cDNA in about 2 hours
  • Compatible with amplified cDNA generated with Tecan’s Ovation® amplification systems
  • Proprietary method eliminates the need for purification steps

The Encore Biotin Module provides optimized reagent mixes and a protocol to process 12 or 60 cDNA samples and is also available in a 96 reaction size for automation workflows.

Applications

  • Gene expression




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Tab 02 / Highlights
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High sensitivity and labeling efficiency

  • High reproducibility and call concordance

Signal correlation for two independent Encore Biotin Module reactions using pooled cDNA generated from 500 pg of HeLa RNA and analyzed on GeneChip arrays, show a high level of signal correlation and call concordance.

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

Amplification Type

Amplification Type

Not applicable

Starting Material

Starting Material

Amplified cDNA

Input Amounts

Input Amounts

2 – 6 ug




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Tab 04 / Faq
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FAQ

Getting started

What equipment is required or will be useful?

Required equipment includes a microcentrifuge, pipettes, vortexer, a thermal cycler, and a Nanodrop or UV/Vis spectrophotometer. An Agilent Bioanalyzer or a similar instrument may be used for quality control.

What additional reagents are required for the Encore Biotin Module?

No additional reagents are required.

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 Technical Report on Encore Biotin Module Performance for a summary of our performance data.

Input recommendations

How should I QC my cDNA for use with the Encore Biotin Module?

You must use cDNA generated with a validated Tecan amplification system. The concentration of starting cDNA must be determined to ensure adequate input into the labeling reaction and, therefore, onto the arrays. Please refer to Table 2 in the User Guide for cDNA input requirements

Can I vary the amount of cDNA input used for fragmentation and labeling?

You may use from 2 to 6 μg of cDNA input with the Encore Biotin Module. Refer to Table 2 in the User Guide to determine the specific recommendations for your sample type, array type and amplification kit used. We recommend using the same cDNA input across all samples in a single experiment where a range of inputs is given.

Can I use any cDNA as starting material in the Encore Biotin Module?

No, the cDNA must be generated using a compatible Tecan RNA Amplification System. Use of other cDNAs will result in poor performance.

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.

cDNA quantification and qualification

How much fragmented and labeled cDNA yield can I expect?

Since this module does not require any purification, the final yield is equal to the amount of cDNA input.

What is the size range of fragmented cDNA 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.

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

Should I purify the labeled cDNA before hybridization?

No. Purification of the labeled cDNA is not necessary.

What types of arrays work with the Encore Biotin Module cDNA?

The Encore Biotin Module has been validated on Thermo Fisher (formerly Affymetrix) 3’ Expression and GeneChip Whole Transcript Arrays (such as Gene ST or Exon ST arrays).

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

No. Refer to Appendix A in the User Guide for information on array hybridization protocols.

What hybridization and wash protocols do you recommend for Thermo Fisher (formerly Affymetrix) GeneChip applications?

Refer to Appendix A in the User Guide for information on array hybridization protocols.

How much labeled cDNA should I hybridize to a GeneChip array?

Refer to Appendix A in the User Guide for guidance regarding the use of the Encore Biotin Module with Thermo Fisher (formerly Affymetrix) GeneChip Arrays.




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Tab 05 / Literature
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Literature

Type
Title
No
Technical Report
Encore Biotin Module
M01311 V1.2
User Guide
M01111 V8.1
Quick Protocol
Encore Biotin Module
M01121 V3.1
Safety Data Sheet
 
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For research use only. Not for use in diagnostic procedures.
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