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 miRNA Microarray. In a single experiment, we currently profile:
- 2,549 human miRNAs represented on the Human miRNA Microarray (Release 21.0)
- 1,881 mouse miRNAs represented on the Mouse miRNA Microarray (Release 21.0)
Tissue, RNA preparation and storage
The storage and preparation of miRNAs from samples are crucial for microarray gene expression analysis. Recently, it has been shown that miRNAs within tissues can be kept in a satisfactory state for prolonged periods using formalin. Xi et al. (2007) found that the expression of miRNAs from formalin-fixed paraffin-embedded (FFPE) specimens was in good correlation with fresh frozen samples, raising the possibility of archiving fixed clinical specimens for further miRNA analysis at a later date. The first step in obtaining miRNA samples is the isolation of RNA from different cells and tissues. Many different in-house protocols and commercial kits can be used. At GPCF we have gathered experience with acidic phenol methods (home-made and commercial (TRIzol, Invitrogen) as well as using silica-based membrane-spin column (miRNeasy, Qiagen). These methods can provide high quality RNA for further investigations.
Although the relative abundance of small RNAs in a total RNA preparation is typically in the range of 0.01-0.05%, it is important that all RNA molecule types are isolated in a single step â€“ no small RNA enrichment should be done! The sensitivity of miRNA detection is ensured by applying an amplification step before hybridization.
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 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/Âµl. You should complete the miRNA profiling sample submission form, 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 (see example expression data)
- 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
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.