Label IT® miRNA Labeling Kits

Rapid and sensitive non-enzymatic labeling of microRNA (miRNA) for microarray analysis

• Sensitive—Confidently detect subfemtomole amounts of miRNA species.
• Universal—Labels miRNAs from all organisms including plants.
• Reproducible—Generate consistent, high quality miRNA microarray data.
• Complete—Single reagent; no additional reagents (dyes) required.
• One-step Labeling Reaction—Rapid and simple direct labeling of miRNA samples.

Validated with the Following Arrays:

• FlexmiR Human Panel - Luminex
• Species specific MicroRNA 4 x 2K Microarrays - Combimatrix
• NCode™ Multi-Species miRNA Microarrays - Invitrogen
• mirVana™ miRNA Bioarrays – Ambion
• miRMAX™ miRNA Microarray X-Species Arrays - Rutgers University

Kit Components:

Each kit includes the Label IT® miRNA Cy™3 and Cy™5 Reagents and additional buffers and reagents required for labeling reactions. This kit also contains a 1X Hybridization Solution. Reagents required for sample preparation and sample purification are not included.

Data

Figure 1: The Label IT® miRNA Labeling Kit Protocol.
Figure 2: Sensitivity of miRNA Microarray Detection.
Figure 3: miRNA Expression Profiling Using the Label IT® miRNA Labeling Kit.

Figure 1. The Label IT® miRNA Labeling Kit protocol. The Label IT labeling reagent comprises a reactive alkylating agent with strong nucleic acid binding capability facilitated via electrostatic interactions. Labeling via covalent modification (alkyation) of RNA (including miRNA) or DNA can take place on reactive heteroatoms on any nucleotide of the nucleic acid polymer. The label does not impact hybridization performance in subsequent applications.

Figure 2. Sensitivity of miRNA Microarray Detection. A chemically synthesized miR-124a RNA oligo was labeled with either Cy™3 or Cy™5 using the Label IT® miRNA Labeling Kit. Defined quantities (0, 0.08, 0.4, and 2 fmol) of each labeled miRNA were spiked into aliquots of pooled Cy™5- and Cy™3-labeled heart miRNA-enriched small RNA samples which are known not to contain the miR-124a miRNA. MicroRNA-enriched small RNA samples were prepared from mouse heart using the mirVana™ miRNA Isolation Kit (Ambion) and labeled with either Cy™3 or Cy™5 using the Label IT® miRNA Labeling Kit. These samples were hybridized to duplicate miRMAX microarrays (Bionomics Research & Technology Center, Rutgers University). The average Cy™3 and Cy™5 signals at the miR-124a features were determined and plotted in comparison to the average Cy™3 and Cy™5 background signals surrounding the miR-124a features in each hybridization.

Figure 3. miRNA Expression Profiling Using the Label IT® miRNA Labeling Kit. MicroRNA-enriched small RNA samples were prepared from mouse brain and heart using the mirVana™ miRNA Isolation Kit (Ambion) and labeled with Cy™3 (brain) or Cy™5 (heart) using the Label IT® miRNA Labeling Kit. The labeled samples were pooled and hybridized to duplicate miRMAX miRNA-specific microarrays. (a) A representative field from a hybridized array. (b) The average corrected Cy™3 (green bars, brain expression) and Cy™5 (red bars, heart expression) signals for the 12 candidate miRNA features. (c) MicroRNA expression patterns determined by northern blotting and published previously (Sempere et. al. 2004. Genome Biology 5(3): R13). The expression pattern for each miRNA is categorized as either “specific” to either of the two organs (H - heart, B - brain) or “enriched” in either of the two organs. In parallel, the miRNA-enriched brain and heart samples were labeled with Cy™3 or Cy™5 using a commercially available miRNA enzymatic labeling kit and hybridized to duplicate miRMAX microarrays. (d) The average M values (log2 [corrected Cy™5 signal/corrected Cy™3 signal]) obtained from both the enzymatically-labeled samples and the Label IT® samples were calculated and plotted. Positive M values indicate greater miRNA expression in the heart compared to the brain, and negative M values indicate greater miRNA expression in the brain than the heart.