Encore^® Biotin Module

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

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

You must use cDNA generated with a validated NuGEN 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. You may choose to further qualify the input cDNA by performing qPCR assays as recommended in the appropriate NuGEN amplification system user guides.

Which protocol should I use for cDNA produced using Applause WT-Amp Plus ST System?

The protocol for labeling cDNA amplified with Applause WT-Amp Plus ST System is found in the User Guide for this product.

What type of cDNA should I use with the Encore Biotin Module?

You must use SPIA cDNA generated with one of the following NuGEN products with the Encore Biotin Module : 

• Ovation RNA Amplification System V2 (Part No. 3100) 
• Ovation Whole Blood Solution (Part Nos. 1300 and 3100) 
• Ovation Pico WTA System V2 (Part No. 3302) 
• Ovation PicoSL WTA System V2 (Part No. 3312) 
• Ovation FFPE WTA System (Part No. 3403) 
• Ovation One-Direct System (Part No. 3500) 
• Applause WT-Amp Plus ST System (Part No. 5510)

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 NuGEN 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.

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.

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.

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 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 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 Affymetrix GeneChip Arrays.