MicroRNAs (miRNAs) are evolutionarily conserved, small, noncoding RNA molecules that negatively regulate gene expression at the level of translation in a variety of eukaryotic organisms. Although the first miRNA was described in 1993, but only recently the breadth and diversity of this gene class been uncovered. Vertebrate genomes encode as many as 1000 unique miRNAs (Berezikov et al., 2006), which are predicted to regulate expression of at least 30% of genes (Lewis et al., 2005).
MicroRNA molecules are produced from larger transcripts that are processed to form hairpin precursors that serve as substrates for Drosha and Dicer, members of the RNase III enzyme family. MicroRNAs are known to regulate the expression of genes involved in the control of development, proliferation, apoptosis, and the stress response (Ambros, 2003 and Chen et al., 2004). Aberrant expression of miRNAs can lead to diseases, including cancer (Calin et al., 2006).
Researchers increasingly seek a global view on the expression of miRNAs in eukaryotic cells and tissue (see 'miRNA Methods' for technical overview). Therefore miRNA profiling on microarrays becomes increasingly popular. However, in contrast to mRNA profiling technologies, miRNA profiling must take into account the difference between mature miRNAs and their precursors, and should also distinguish between miRNAs that differ by as little as a single nucleotide. Moreover, it has been shown that the sequences of mature miRNAs display unequal melting temperatures. These features - specific for miRNAs, present major challenges and require most robust set-ups in order to obtain reproducible data.
We offer a full service of genome-wide miRNA profiling for human and mouse samples, using a well established SOP. GPCF makes use of the Agilent Human miRNA Microarray. In a single experiment, we currently profile:
- 1205 human and 144 human viral miRNAs represented on the Human miRNA Microarray (Release 16.0)
Input from you
As starting material we take total RNA that needs to be prepared in by a protocol that co-isolates small (miRNA, tRNA, 5.8S rRNA) and large RNAs (mRNA, 18S rRNA, 28S rRNA). Do not prepare small RNA separately, as our QC set-up requires mRNA as well as rRNA species. Phenol-based methods (for detailed protocols see WW@DKFZ Chomczynski & Sacchi (1987) or TRIzol, Invitrogen) and dedicated silica-based spin columns (miRNeasy, Qiagen) seem to work fine.
Please provide a minimum amount of 500 ng of total RNA at a concentration of >50 ng/µl. You should complete the miRNA profiling sample submission form (login required), prior to submitting your samples. Please always provide samples sets of 8 samples (or multiple thereof).
Please be aware: NO BIOLOGICAL REPLICATES = NO STATISTICS
What we do
We perform incoming QC for quality and concentration of all samples (Nanodrop ND-1000, Agilent 2100 Bioanalyzer).
Upon acceptation of samples we perform labeling and hybridization to the microarrays, monitoring the quality at all steps.
Image acquisition, single chip analysis as well as normalization across all of your samples is performed.
Output to you
- All RNA and labeling QC data (conc. of input RNA, quality of input RNA, conc. of double stranded cDNA, conc. of labeled cRNA, quality of cRNA)
- Raw data of microarrays
- Individual chip QC (for chip integrity and hybridization success)
- Raw data evaluation (box plots)
- Interpreted data (numerical readout and gene annotation for microarray elements (probe sets)
- Normalization across each chip
- Normalization (if wanted) for complete sets of experiments
- Basic analysis on Excel level
More…
On our miRNA Analysis page you find our detailed technical workflow including all controls. You'll also find a demo analysis of a set of hybridizations, providing some insight in the QC and calculation measures.
For more information, please contact us directly; consult the pricelist for detailed cost information.
