Normalization of microRNA expression levels in quantitative RT-PCR assays: identification of suitable reference RNA targets in normal and cancerous human solid tissues

Abstract

Proper normalization is a critical but often an underappreciated aspect of quantitative gene expression analysis. This study describes the identification and characterization of appropriate reference RNA targets for the normalization of microRNA (miRNA) quantitative RT-PCR data. miRNA microarray data from dozens of normal and disease human tissues revealed ubiquitous and stably expressed normalization candidates for evaluation by qRT-PCR. miR-191 and miR-103, among others, were found to be highly consistent in their expression across 13 normal tissues and five pair of distinct tumor/normal adjacent tissues. These miRNAs were statistically superior to the most commonly used reference RNAs used in miRNA qRT-PCR experiments, such as 5S rRNA, U6 snRNA, or total RNA. The most stable normalizers were also highly conserved across flash-frozen and formalin-fixed paraffin-embedded lung cancer tumor/NAT sample sets, resulting in the confirmation of one well-documented oncomir (let-7a), as well as the identification of novel oncomirs. These findings constitute the first report describing the rigorous normalization of miRNA qRT-PCR data and have important implications for proper experimental design and accurate data interpretation.

FIGURE 1.

Strategy for the identification of stable targets for normalizing microRNA qRT-PCR data.

FIGURE 2.

geNorm and NormFinder analyses of qRT-PCR data from a “horizontal scan” of 13 normal human tissues. (A) Correlation of the geNorm M value and the NormFinder stability value for the 12 RNA targets evaluated. (B) The average standard deviation across all tissue samples when normalized to (1) geometric mean (GeoMean) of miR-191 and miR-93, (2) miR-191, (3) total RNA mass, (4) miR-16, (5) let-7a, (6) U6 snRNA, and (7) 5S rRNA.

FIGURE 3.

Differential expression of hsa-let-7a in human lung cancer: impact of normalizer stability on qRT-PCR expression analyses from both flash-frozen and FFPE tumor and normal adjacent tumor tissues. (A) Hsa-let-7a differential expression in 12 flash-frozen lung cancer NAT pair as normalized to (1) geometric mean (GeoMean) of miR-191 and miR-24, (2) miR-191, (3) miR-103, (4) 5S rRNA, (5) total RNA, and (6) miR-30d. The dotted line indicates the respective average differential expression value. Negative values indicate reduced let-7a expression in lung cancer samples and positive values indicate increased let-7a expression in lung cancer samples. (B) Hsa-let-7a differential expression in 16 FFPE lung cancer NAT pairs as normalized to (1) geometric mean (GeoMean) of miR-17–5p and miR-24, (2) miR-103, (3) miR-191, (4) miR-16, (5) miR-25, and (6) total RNA. The dotted line indicates the respective average differential expression value. (_*) Indicates an outlier. Negative values indicate reduced let-7a expression in lung cancer samples and positive values indicate increased let-7a expression in lung cancer samples.