Finding the Best Endogenous Controls for Real-Time Quantitation of miRNA
|Endogenous controls for quantitating miRNA expression levels using real-time PCR methods should ideally share similar properties with miRNAs (e.g., stability and size) and be amenable to the miRNA assay design. Applied Biosystems scientists performed a systematic study of noncoding RNA species (size range: 45–200 nucleotides) using a variety of tissues and cell lines to determine their suitability as endogenous controls for TaqMan® MicroRNA Assays .|
The Importance of Good Controls
Summary of Methods
Recommendations for Endogenous Controls
- Human: RNU48, RNU44, U47, and RNU6B
- Mouse: snoRNA202 and snoRNA234
- Rat: U87 and 4.5S RNA(H)
Figure 1. Candidate Endogenous Control RNAs for TaqMan® MicroRNA Assays. These human small nuclear RNAs and mouse small nucleolar RNAs were found to have the least variable expression levels when tested in 38 normal tissues (human, orange bars), 59 NCI-60 cell lines (human, green bars), and 12 normal tissues (mouse). Search for publication #127AP11-01 at www.appliedbiosystems.com for more details and data from this study .
Alternate normalization options include using the most stable miRNA(s) in the study or a commonly used endogenous control gene (e.g., 18S rRNA). Regardless of the control gene or gene set that is chosen, we highly recommend that the consistency of expression be reconfirmed under the specific conditions of each experiment.
Linda Wong, Kathy Lee, Iain Russell, Caifu Chen • Applied Biosystems, Foster City, CA