Introduction to GenoExplorer™
miRNA Services
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™ 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.
3. 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.
4. 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.
5. 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.
6. RNA Quality Evaluation for miRNA Experiments
High quality and sufficient concentration of RNA samples
are crucial for the 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.
7. Amount and Concentration of RNA Samples
We use total RNA. The concentration of the RNA samples should
be >0.5 µg/µl in DNase/RNase-free H2O. We usually
require 1-2.5 µg of total RNA for each testing sample(s)
to achieve optimal subfemtamole sensitivity, although 100-200
ng total RNA can generate biologically significant results.
If you do size fractionation (although it is not required),
fractionated RNA (proportion of 1-2.5 µg of total RNA)
dissolved in 10 µl RNase-free H2O is required. If you
are limited in sample amount, please call us to discuss options
of accommodating smaller sample sizes.
8. miRNA Labeling and Expression Profiling Technology
Total RNA in the amount of 1-2.5 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.
9. miRNA Detection
Array chips are scanned and fluorescent signals are captured
for microRNA detection. The signal intensity and background
are computed. miRNA signals are normalized using control genes.
The analyzed data is stored and exported to an application
program for further analysis.
10. Submitting Your Samples
Before you submit your samples, please make sure 1) The RNA
quality has been properly evaluated. An agarose gel image
of the RNA samples is required. 2) Total RNA for each sample
should be no less than 2.5 µg in the concentration of
>0.5 µg/µl in DNase/RNase-free H2O. 3) Samples
should be sent on dry ice via an overnight courier to the
company's address below. All RNA samples are stored in -70°C
upon arrival. 4) Complete the Sample Testing Submission Form
with the appropriate information.
Testing Samples Receivable
GenoSensor
4665 S. Ash Ave. Suite G-18
Tempe, Arizona 85282
USA
11. Final Report
We will send you the data in a compact disc that contains
all scanned chip images in tif image format, and raw data
for each sample in an Excel spreadsheet. The raw data is gridded
and computed by standard microarray analysis software, which
is easily transferable to other programs for further analysis.
A data analysis report is also provided. This data summary
includes all gene names, fluorescent signal intensity values
of each probe, mean value of triplicate probes, standard deviations,
gene normalization, and fold changes between samples. The
chip layout that provides with the genes and their array positions
on the chips is also included on the CD.
12. Turnaround Time
Ensuring quality of service and shortening turnaround time
are our primary concerns. The turnaround time normally is
one week to 10 days depending on the number of processed samples
and order that samples were received.
13. Product and Service 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
Back to GenoExplorer™ microRNA
Expression Analysis |
