Procedures
Tissue
isolation and RNA Preparation:
Tissue Isolation and RNA
Preparation: Surgical specimens are emerged in liquid nitrogen upon
obtaining and either used immediately or stored in - 80oC. Each tissue
is divided into matched fractions for RNA isolation and histology.
Total RNA is prepared using Trizol. Back to top
Histology:
Samples are fixed at room
temperature in neutral pH phosphate-buffered 10% formalin, dehydrated
in graded alcohols, and embedded in paraffin using an automated tissue
processor. Four mm thick paraffin sections are rehydrated and stained
routinely with hematoxylin and eosin in a diagnostic pathology laboratory.
Photomicrographs of representative areas for each accession are prepared
from these slides. Back to top
Biotin
labeling of the RNA:
Total RNA is converted
to biotin labeled cRNA using standard biochemical procedures (see
Wodicka et al Nature Biotechnology 15:1359-1367, 1997). Briefly, 7-10
mg of total RNA is reverse transcribed using an oligo-dT primer linked
to a T7 polymerase recognition site. Double stranded cDNA is synthesized
and used for an in vitro transcription reaction in the presence of
biotin-dUTP. The amplified cRNA is purified and quantitated. Back
to top
Hybridization
and scanning:
cRNA is fragmented by incubation
at 94oC for 30 minutes in the presence of 40 mM Tris-acetate, pH 8.1,
100 mM potassium acetate, and 30 mM magnesium acetate. Fragmented
cRNA (20 mg) is hybridized to the GeneChip. Back to
top
Quality Assurance:
Prior to hybridization
to experimental arrays, the quality of cRNA was assessed using test
arrays (Affymetrix Test2 gene arrays) designed to compare relative
expression levels of (-actin and GAPDH by using oligonucleotide probes
complementary to both the 3' and 5' ends of gene products. Data failed
to meet the criteria of 3'/5' ratio less than 3 according to the manufacturer's
instructions were excluded from analysis. Additionally, the quality
of the samples was assessed by spiking the test sample with a known
amount of prokaryotic control cRNA. Each probe array contains probes
for several prokaryotic genes that serve as a hybridization control
by allowing the user to equate a known amount of cRNA with a measured
expression level.Back to top
Data
Analysis:
GeneChip(r) Software (Affymetrix
Microarray Suite 4.0 (r)) was used to generate quantitative gene expression
values measured by the "average difference" between the hybridization
intensity with the perfect match (PM) probe sets and the mismatch
(MM) probe sets. The raw expression levels were then multiplied by
a scaling factor to make the mean expression level on the microarray
equal to a "target intensity" of 100. This scaling is automatically
performed by the Affymetrix software to normalize the gene expression
levels to allow comparison between any two samples.Back
to top
How
to search:
The HuGE database contains
gene expression levels of 6000 human genes determined using the Affymetrix
HuFL GeneChip(R) probe array. This particular GeneChip probe array
contains primarily known genes, i.e., those that have been previously
characterized in terms of function or disease association. There are
very few orphan ESTs.
This on-line search engine
is a simple interface that allows you to query the database to identify
which mRNAs are expressed in a variety of normal tissues and determine
their relative levels of expression. If you are interested in performing
a more rigorous analysis of the data, please download data of interest
(copy and paste) and use your own software tools. Back
to top
Searching:
The search engine can operate
in one of two modes; it will search using either a specific term that
you enter OR it will search specific tissue(s) that you choose from
a list. Currently, you cannot simultaneously search a term and a selected
tissue. Search using a text term: Using the text box at the top left
of the page, enter a term. The search term can be general (e.g., kinase)
or relatively specific (e.g., actin). The engine will search the entire
database of all tissues for this term. Currently, it is very permissive
and finds all identical matches to the term. Do not select a tissue
when searching with a term. Search by tissue(s): In this mode you
select one or more tissues by clicking on the tissue name(s) shown
in the box on the top right of the page. Hold down the Control button
to select more than one tissue. Do not enter a text term when searching
specific tissue(s). Back to top
Display
Options:
Present, Absent, Marginal,
All Genes: In any given tissue only 20-30% of the genes are generally
detected as being expressed. Thus, to limit the number of genes that
are retrieved in a search you can choose to view only those genes
that were found to be present. Alternatively, you may wish to see
all the genes, or only those that are absent or marginal. Select All
Genes if you wish to obtain a list of all the genes that are on the
chip.
Display Order: Alphabetical,
ascending expression level, descending expression level. The search
engine will sort and display the data in any of these three modes.
When comparing different tissues, alphabetical sorting is most useful
as it makes it easier to compare the same gene in several tissues.
Average Value or Individual
Samples: For many tissues we have obtained more than one sample.
You can choose to view either the average value of the expression
level for all the samples or you can view the expression levels of
all the individual samples.
Attributes: Each
data point in the database has a variety of attributes that will assist
you in identifying genes of interest. These can be changed to fit
your needs. The gene function attribute is not currently enabled as
it is still under development. Back to top
Copy
and Paste:
When the desired data are
displayed, the user may import the dataset via the copy and paste
function of the web browser onto his/her own computer. In order to
do this, highlight the desired cells by holding the left mouse button,
then choose the "copy" function from the web browser's Edit menu.
The information can then be "pasted" into a spreadsheet or used as
a tab-delimited text file. Note: Hyperlinks can be maintained if the
recipient program allows this feature.Back to top
Current
Limitations and Future Plans:
The search engine and database
are still under development so the search capabilities are somewhat
limited. In the future, the search engine will be more flexible allowing
one to enter a search term and also select a limited number of tissue
samples. In addition, it will be possible to search multiple terms
or genes at once. The functional annotation of the genes has not been
completed and will be completed in the future. Back
to to
FAQ