Analysis of miRNA Expression Profiles
MicroRNAs (miRNAs) are a large class of small non-coding RNAs (21-25 nucleotides) that regulate gene expression at post-transcriptional level, inducing degradation of specific RNA messengers (mRNAs), or preventing translation into proteins. Analysis of miRNA expression profiles, by microarray, allows to simultaneously evaluate the expression of hundreds of miRNAs in a biological sample. The principle on which it is based is that of hybridization between labeled miRNA molecules and thousands of specific oligonucleotides linked to a glass support. Numerous studies have demonstrated the involvement of miRNAs in various cardiovascular, neurological, obesity, diabetes and cancer pathologies. miRNAs have biological characteristics that make them potentially suitable for clinical applications such as early diagnosis markers and monitoring of therapeutic strategies.
Microgem Lab provides the one-color, miRNoma extended coverage, “Analysis of miRNA Expression Profiles” service with high density microarrays, high sensitivity even for low expression levels, very high accuracy. Possibility to analyze miRNAs extracted from tissues and cells of a wide variety of organisms and species.
Workflow of one-color “Analysis of miRNA expression profiles” service
- Consultancy – A free consultation will be provided for project optimization (number of samples to be analyzed, methodology, costs).During the consultation phase, advice will be provided on the extraction method to be taken. Alternatively, you can take advantage of our experience in RNA isolation.
- Sample Requirements –
- 2 ug of total RNA, from tissues, cells, plasma, serum, of human, mouse, rat, at concentration over 50 ng/ul
- RNA samples must undergo to DNase I treatment
- RNA samples are required to have an OD 260/280 ratio between 1.8-2.0 and 260/230 ratio ≥7
Please supply a shot of 1.5 % denaturating agarose gel.
- Sample Quality Control (QC) - After receiving RNA samples, our researchers will evaluate the integrity and purity of each sample by MaestroNano microvolume reader and Bioanalyzer. Possible contamination for each sample will also be assessed. The results of this analysis will be sent before proceeding with the other phases of the service.
- Labeling, Hybridization and Reading of the Microarray – The RNA sample will be dephosphorylated, labeled with Cyanine3-pCp and hybridized on specific microarray. For workflow monitoring, Spike-In controls (Drosophila miRNA) will be inserted into each sample during the labeling procedure and a specific RNA reference will be used in a parallel experiment. Additionally, negative and positive controls for self-monitoring and cross-hybridization are stored on each microarray. After washing, the microarray will be analyzed by laser scanner and the fluorescence image will be digitized.
- Data Analysis – Data quality analysis based on Spike-In controls, spot intensity and flagged spots. Comparison of data obtained from independent microarrays will be normalized. A Principal Component Analysis (PCA) will be performed to identify variations related to the biological sample or technical reasons.
- Statistical Data Analysis – Next, statistical data analysis will be performed for the determination of differently expressed miRNAs (T-test or Anova for determination of p-values, with or without correction, MA and Box plot). A Volcano plot analysis will allow you to display miRNAs with the lowest p-values. A Heat Map plot will display the data of an unsupervised hierarchical clustering of miRNAs on all analyzed samples. Additionally, a prediction of molecular targeting of miRNAs by database (TargetScan, TarBase) will be performed.
- Final Report – At the end of the project, a final report will be provided including the results of the various stages of project development. The results presented in the report can be discussed with the scientific team.