RNA Isolation Techniques
****All total RNA samples submitted to the Duke Microarray Facility must be in nuclease-free water.****
To achieve high quality RNA samples we recommend extracting with Qiagen’s RNeasy kit (or a comparable product). We recommend that the optional DNase treatment be performed to eliminate genomic DNA contamination.
If you choose to use Trizol or another phenol/chloroform extraction method, we strongly recommend that you perform a column cleanup of the sample. Residual organics can severely compromise RNA labeling and amplification (if needed) and negatively affect array results.
Researchers should be aware that RNA extraction using beta-mercaptoethanol (typically for plant cells) will cause high background on Affymetrix GeneChip arrays and potentially confound the resulting data.
Special cases:
- miRNA assays: DO NOT enrich the sample for small RNA. Use a column that does not exclude miRNA and yields total RNA. Ambion’s miRVANA Total RNA Isolation kit is a good choice.
- Blood samples: Globin reduction must be performed on RNA extracted from blood. Otherwise, the globin will be amplified, labeled and hybridized along with the RNA sample, adversely affecting array results and yielding misleading data.