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
Quantitation of miRNA expression levels is essential for understanding their roles in cellular processes (e.g., proliferation, differentiation, and death) and identifying potential biomarkers for human diseases. Normalization with endogenous control genes is currently the most accurate method to correct biases from variations in RNA input or reverse transcription efficiency when measuring miRNA expression. The expression pattern of an ideal endogenous control demonstrates relatively constant and highly abundant levels across tissues and cell types; however, no single control can serve as a universal endogenous control for all experimental conditions.
Summary of Methods
TaqMan Assays were designed to target 38 human small nuclear RNA (snRNA), small nucleolar RNA (snoRNA), and transfer RNA (tRNA). After a preliminary screen using human lung total RNA, 23 assays for snRNAs and snoRNAs were further tested as potential control assays using RNA from 38 normal human tissues and 59 NCI-60 cell lines. A similar, but smaller, set of experiments was performed to identify mouse and rat endogenous control candidates.
Recommendations for Endogenous Controls
A set of endogenous RNAs (18 in human, ten in mouse, and five in rat) were identified as good candidate controls for normalizing miRNA gene expression data [1, and unpublished results]. The results of this study indicated that the following snRNA/snoRNA endogenous controls have the highest abundance and least variability across the normal tissues and cell lines that were tested (Figure 1):
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