Microarray support

Sample isolation and input recommendations

RNA input recommendations

RNA isolation

We recommend a column-based method, including:

Note: Do not use carriers during RNA isolation. Organic methods such as TRIzol® Reagent should be subsequently followed with a column-based clean-up method.

cDNA clean-up

cDNA generated with NuGEN cDNA Systems should be cleaned up using a column-based method such as:

Alternatively, an SPRI bead-based system such as Agencourt® RNAClean® XP or AMPure XP Systems are suitable. If choosing a bead-based method, we recommend using a magnet compatible with 0.2 mL tubes, tube strips, or plates.

Note: Beads for final cDNA cleanup must be purchased separately from Beckman Coulter.

Fragmentation, Labeling, and Hybridization of cDNA for Microarrays

Fragmentation and labeling for Affymetrix Microarrays

NUGEN AMPLIFICATION SYSTEM (Part No.) cDNA INPUT PER REACTION* FINAL HYB COCKTAIL CONCENTRATION
Ovation One-Direct RNA Amplification System (Cat. #3500) 5–6 μg 23–27 ng/μL
Ovation FFPE WTA System (Part No. 3403) 4-5 μg 18–23 ng/μL
Ovation Pico WTA System V2 (Part No. 3302) 5 μg 23 ng/μL
Ovation PicoSL WTA System V2 (Part No. 3312) 2.5 μg** 23 ng/μL
Ovation RNA Amplification System V2 (Part No. 3100) 3.75 μg 17 ng/μL
Ovation Whole Blood Solution (Part No. 1300 & 3100) 4.4 μg 20 ng/μL
Applause WT-Amp Plus ST System (Part No. 5510) 4–5 μg 18–23 ng/μL

* All cDNA concentrations are assessed using 33 μg/mL/A260 as the constant.

** Requires performing half-volume Encore Biotin Module reactions. Refer to the appropriate Ovation PicoSL WTA System user guide for specific guidelines.

Hybridization of Labeled cDNA Target on Affymetrix Microarrays

Hybridization, cocktail assembly and fluidics protocols for single GeneChip® Arrays using Affymetrix HWS kit (Affymetrix P/N 900720)

Component Standard Array (49 or 64 Format) Midi Array (100 Format) Mini Array (169 Format) Final Concentration
Fragmented, biotin-labeled amplified cDNA 50 μL 34 μL 25 μL Depends on sample type and amplification method
Control oligonucleotide B2 (3 nM) 3.7 μL 2.5 μL 1.9 μL 50 pM
20X Eukaryotic hybridization controls (bioB, bioC, bioD, cre) 11 μL 7.5 μL 5.5 μL 1.5, 5, 25 and 100 pM, respectively
2X Hybridization buffer 110 μL 75 μL 55 μL 1X
100% DMSO 22 μL 15 μL 11 μL 10%
Water 23.3 μL 16 μL 11.6 μL N/A
Total Volume 220 μL 150 μL 110 μL  
Array Loading Volume 200 μL 130 μL 90 μL  
FLUIDICS PROTOCOLS
For 3’ arrays FS450_0004 FS450_0002    
For ST arrays FS450_0001 (Exon arrays)   FS450_0007(Gene arrays)  

* All cDNA concentrations are assessed using 33 μg/mL/A260 as the constant.

** Requires performing half-volume Encore Biotin Module reactions. Refer to the appropriate Ovation PicoSL WTA System user guide for specific guidelines.

Fragmentation and labeling for Illumina BeadArrays

Two to four micrograms of amplified and purified cDNA from one of the compatible amplification kits is required as input into the labeling reaction. The amount of cDNA to label depends on the BeadChip type used. We have observed consistent and good results using 750 ng to 1.5 μg labeled target in the final hybridization cocktail.

Hybridization of Labeled cDNA Target on Illumina BeadArrays

NuGEN recommends hybridizing approximately 750 ng to 1.5 μg of labeled cDNA for all of the Illumina Whole Genome Expression BeadChips. Hybridization cocktails should be prepared and array hybridization and wash conditions followed essentially as recommended by the array manufacturer with the following exception: NuGEN recommends hybridizing cDNA targets at a reduced hybridization temperature of 48°C.

NuGEN’s Encore Biotin and Encore Biotin IL Modules are designed solely for use with cDNA prepared using following NuGEN kits:

  • Ovation® RNA Amplification System V2 (Part No. 3100)
  • Ovation Whole Blood Solution (Part Nos. 3100 and 1300)
  • 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) (Encore Biotin Module only)

*Note: With the Ovation PicoSL System V2, use half the recommended volumes throughout the Encore Biotin Labeling protocol.