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

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.

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 μg23–27 ng/μL
Ovation FFPE WTA System (Part No. 3403)4-5 μg18–23 ng/μL
Ovation Pico WTA System V2 (Part No. 3302)5 μg23 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 μg17 ng/μL
Ovation Whole Blood Solution (Part No. 1300 & 3100)4.4 μg20 ng/μL
Applause WT-Amp Plus ST System (Part No. 5510)4–5 μg18–23 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)

ComponentStandard Array (49 or 64 Format)Midi Array (100 Format)Mini Array (169 Format)Final Concentration
Fragmented, biotin-labeled amplified cDNA50 μL34 μL25 μLDepends on sample type and amplification method
Control oligonucleotide B2 (3 nM)3.7 μL2.5 μL1.9 μL50 pM
20X Eukaryotic hybridization controls (bioB, bioC, bioD, cre)11 μL7.5 μL5.5 μL1.5, 5, 25 and 100 pM, respectively
2X Hybridization buffer110 μL75 μL55 μL1X
100% DMSO22 μL15 μL11 μL10%
Water23.3 μL16 μL11.6 μLN/A
Total Volume220 μL150 μL110 μL 
Array Loading Volume200 μL130 μL90 μL 
FLUIDICS PROTOCOLS
For 3’ arraysFS450_0004FS450_0002  
For ST arraysFS450_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.