Overiew of microRNA
GenoExplorer™ microRNA Products
Facility and Environment of Fabrication and Laboratory
GenoExplorer™ miRNA Biochip (microarray) products and services are manufactured and performed in class 100 cleanroom facilities under stringent GMP and GLP protocols.
GenoExplorer™ microRNA Products
1 Full Kit Components
GenoExplorer™ miRNA array chips
GenoExplorer™ miRNA labeling kit containing labeling enzyme and mix
Hybridization buffer
Washing solutions
User’s manual
2.2 Separate Components are also available
GenoExplorer™ Biochip Platform
GenoExplorer™ Biochips are established on a 25×75 mm high-quality glass microslide with a 3D matrix. The DNA oligo probes are synthesized, and then immobilized onto the chips. Only the full-length probes attach to the chips, resulting in a high-quality performance indicated by high reproducibility, signal to noise ratio, and sensitivity. Scanners capture the fluorescent signals generated from the hybridization assays.
microRNA Array Design
GenoExplorer™ microRNA chips contain the most comprehensive probe content for microRNA profiling collected from all public domains. Nomenclature is based on Sanger miRNA registry. The genes on the chips are kept updated. Chips are manufactured based on the species, such as human, mouse, etc. Probe designs have been computed and optimized based on their hybridization thermodynamics. The chips offer the user the opportunity to perform differential expression analyses between different samples, not only for the mature miRNA, but also for the precursor miRNA. Multiple positive and negative controls are included on the chips such as U6, 5S rRNA, and tRNAs, which also serve as loading controls. Negative controls are used for chip background. Each probe is printed in triplicate. The GenoExplorer™ microRNA chips also include multiple probes for external RNA controls. The external RNA controls, which are included in the labeling kit, can be added to the total RNA at the beginning of the labeling protocol. The controls enable the user to evaluate the quality of the labeling and hybridization steps. The control RNA sequences have been validation tested to ensure they do not cross-hybridize to any of the microRNA probes on the chip
Types of GenoExplorer microRNA Biochips
Chips are designed based on specific species and available for all species.
GenoExplorer™ microRNA – Human
GenoExplorer™ microRNA – Mouse
GenoExplorer™ microRNA – Other species including rat, Drosophila, C elegans, and Arabidopsis thaliana are available.
Isolation of Total RNA for Array Experiments
The main goal is to obtain good quality and sufficient amount of RNA. Many laboratories use the Trizol reagent for isolation which is recommended. However, each sample may result in different quality, and further evaluation on those samples is necessary. RNA size fractionation is not required for high integrity of RNA samples
RNA Quality Evaluation for miRNA Experiments
High quality and sufficient concentration of RNA samples are crucial for experiments on microarrays. RNA quality can be evaluated by visualizing the RNA on a gel, as well as by calculating the A260/A280 ratio. On a denaturing gel (or on an ordinary agarose gel in denaturing buffer) the RNA should appear as two bright distinct bands that represent the 28S and 18S ribosomal species. The 28S band should be brighter than the 18S band. Tailing of these major bands down the gel, or a background smear behind these bands that gets heavier at lower molecular weights can indicate degradation of the RNA. Degraded RNA will produce high background and low signal intensities on microarrays. The presence of very sharp bands higher than the 28S ribosomal band can indicate the presence of excess DNA in the sample, which can be removed by treatment with RNase-free DNase I. The spectrophotometric ratio will also give an indication of the purity of the RNA, and should be as close to 2.0 as possible. Generally, ratios less than 1.7 indicate that the RNA may be contaminated with other material and should be repurified, or perhaps run through a column
Amount and Concentration of RNA
We use total RNA. The amount of RNA sample should be >1 µg at 0.5 µg/µl in DNase/RNase-free H2O. We usually use 1-2.5 µg of total RNA for each testing sample(s). If you do size fractionation (although it is not required), fractionated RNA (proportion of 1-2.5 µg of total RNA) dissolved in 10 µg RNase-free H2O is required.
miRNA Labeling and Expression Profiling Technology
Total RNA in the amount of 10 µg per sample is required for the assay. RNA is isolated from tissues and cells using traditional methodology, such as Trizol method. The GenoExplorer™ microRNA labeling technology offers a highly selective and uniform labeling system and a rapid labeling protocol. The labeling is uniform, ensuring that the RNA reacts with a single label. Finally, the rapid labeling protocol allows the user to complete the labeling and begin the hybridization on the same day. The labeled RNA is then used for the on-chip hybridization assays under optimized conditions. The fluorescent signals from hybridized targets are scanned and analyzed
Product Features
- Labeling – direct, minimal labeling bias, fluorescence-based detection
- Platform – flexible and compatible with most hybridization chambers and microarray scanners
- Sensitivity – detects down to subfemtamole levels starting with 1 µg of input total RNA
- Specificity – discriminates sequences with up to 94% homology
- Differential Expression Detection – enables detection of less than 2-fold differential miRNA expression
- Reproducibility – CV < 15 % between experiments
- Dynamic Range – > 3 logs of dynamic range