Amplification Overview

MicroRNA expression profiling on microarray platforms generally requires at least 5 ug of starting total RNA. If your research involves working with special cell types, tissues, or serum, obtaining 5 ug of total RNA might be challenging. ORB miRNA amplification system is a sensitive and efficient system for amplifying microRNA from as low as 50 ng of total RNA or 10 ng enriched low molecular weight RNA (LMW). Designed based on Goff et al. (2004), ORB microRNA amplification system provides at least 1000 fold amplification of LMW RNA. ORB microRNA amplification system maintains the relative levels of microRNAs during the amplification process.

Performance Data

100 ng of RNA from a pool of total RNA from 20 different human tissues (Universal Pool) was amplified by ORB miRNA amplification system in duplicate. 200 ng of enriched LMW RNA from the same source was labeled with the NCode™ miRNA Labeling System (Invitrogen) without amplification. The amplification was highly reproducible with little to no variation for the detectable probes. (See figure 1 & 2 below)

Figure 1:Reproducibility plot for amplified total RNA

Amplification Schema

Figure 2:Reproducibility plot for directly labeled low molecular weight RNA

Agreement of differential microRNA expression between amplified and directly labeled samples

100 ng aliquots of total RNA from Human Kidney or Human Universal Pool were amplified using the ORB miRNA Amplification System and biotin labeled for array analysis. Additionally, 200 ng aliquots of enriched LMW RNA prepared from the same sources were directly labeled with the NCode™ miRNA Labeling System (without amplification) and each were hybridized to ORB Sanger 11 miRNA microarrays. MicroRNAs were selected for further analysis if they were detectable on arrays hybridized with both amplified and unamplified samples and showed at least 2-fold differential expression between Universal Pool and Kidney. 93% of the microRNA examined showed similar differential expression in the amplified samples vs. the directly labeled samples (Fig. 3A and 3B).

Figure 3A:Agreement of differential MicroRNA expression between amplified and directly labeled samples

Figure 3B: Agreement of differential MicroRNA expression between amplified and directly labeled samples (continued)

MicroRNA expression profiling in FFPE samples

MicroRNAs are not significantly affected by the formalin fixation process used in preparing Formalin-Fixed, Paraffin-Embedded (FFPE) tissue samples. To exploit the opportunity of using archived samples to detect changes in miRNA expression levels in various disease states, we offer microRNA profiling from archived FFPE tissue samples. We isolate total RNA from as little as 8 sections (20 microns each) using Ambion's Recover all Total Nucleic Acid Isolation Kit, and enrich the low molecular weight RNA in the samples. Samples are then labeled using NCode™ miRNA Labeling System and hybridized to ORB Sanger 13 Multispecies array (Human, Mouse or Rat). The resulting FFPE microRNA expression profiles correlates well with that of microRNA expression profiles from fresh tissues.