Introduction to GenoExplorer™ miRNA
1. 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.
2. GenoExplorer™ microRNA Products
2.1 Full Kit Components
GenoExplorer™ miRNA array chips
GenoExplorer™ miRNA labeling kit containing labeling
enzyme and mix
2.2 Separate Components are also available
3. GenoExplorer™ Biochip Platform
GenoExplorer™ Biochips are established on a 25x75 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.
4. 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.
5. 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.
6. 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.
7. 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.
8. Amount and Concentration of RNA Samples
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.
9. 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.
1 0. Product Features
Labeling - direct, minimal labeling bias, fluorescence-based
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
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