Microarray support
Sample isolation and input recommendations
RNA input recommendations
- Total or poly A selected RNA
- High quality, with an A260:A280 ratio of 1.8 or above and a RIN score above 7
- DNase treated. We recommend a DNase such as HL-dsDNase from ArcticZymes. Other suitable DNase treatments include the Qiagen RNase-free DNase included with the RNeasy Mini RNA Purification and RNeasy Micro Purification Kits, or Roche RNase-free DNase followed by clean-up with Zymo RNA Clean & Concentrator 5 or Qiagen RNeasy MinElute Columns. Note that column-based methods may not provide sufficient removal of DNA.
- While high quality RNA will guarantee the best results, many of our kits will perform with degraded samples.
RNA isolation
We recommend a column-based method, including:
- Norgen Biotek Total RNA Purification Kit
- Zymo Research QuickRNA TM Kits
- Arcturus PicoPure® RNA Isolation Kit
- Ambion PureLink® RNA Mini Kit
- Qiagen RNeasy Kits
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:
- QIAGEN MinElute Reaction Cleanup Kit
- QIAGEN QIAquick PCR Purification Kit
- Zymo Research DNA Clean & Concentrator TM -25
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
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.
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.