Complete and Accurate miRNA Expression Profiles – Detect miRNAs Missed by Enzymatic miRNA Labeling Kits

Discrepant microarray miRNA profiles are obtained from chemical and enzymatic labeling methods. Only the miRNA expression profile generated using the Label IT® method corroborates the established expression pattern obtained by northern blotting.* miRNA-enriched mouse heart and brain samples were hybridized to miRNA microarrays after labeling with either Label IT®, a direct chemical labeling method, or mirVana™ (Ambion) or NCode™ v.1 (Invitrogen) enzymatic methods. Positive M values, expressed as the log2 transformed ratio of heart/brain signal, represent mouse miRNAs preferentially expressed in heart tissue, while negative values correspond to miRNAs preferentially expressed in brain.

*Sempere, L.F., S. Freemantle, I. Pitha-Rowe, E. Moss, and V. Ambros. (2004) Genome Biol. 5:R13

(A) Synthetic miRNAs representing positive controls and test miRNAs (10 fmol each) were spiked into the appropriate miRNA-enriched samples lacking each miRNA. The spiked samples were then labeled either chemically or enzymatically using the indicated kits and hybridized. (B) Synthetic positive control miRNAs were consistently detected on microarrays using all three labeling methods. Synthetic test miRNAs were only detected after labeling with the Label IT® miRNA Labeling Kit.

Direct chemical labeling using the Label IT® miRNA Labeling Kits maintains the integrity of the original miRNA species. Only the fluorophore and Label IT® chemical linker are added to the miRNA. The poly(A) polymerase-based mirVana™ and NCode™ Labeling Kits add approximately 200 to 300% extraneous nucleotides to the 3’ end of each miRNA. The poly(A) polymerase-based methods may not label all miRNAs or the additional nucleotides may adversely affect hybridization performance.

The Label IT® miRNA Labeling Reagents are composed of a fluorophore (or biotin) attached to the positively charged Label IT® linker and reactive alkylating group. The Label IT® Reagents covalently attach fluorophores (or biotin) to any reactive heteroatom in miRNAs without affecting downstream hybridization performance. miRNA labeling and purification can be completed in only two hours.