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HugeIndex Procedures

Contents


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.

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.

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.

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.

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.

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.

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.

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).

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.

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.

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.

Protein Engineering