Laboratory Services

New Sample Drop-off Room Information

The Sample Drop-off room for the Microarray Core is located in Smith, room 1129. Please continue to send a completed application form to Edward Fox (Edward_Fox@dfci.harvard.edu). You will need a printed copy of the application form as well to include with your samples. For RNA samples, place your 1.5mL tubes (with the sample name written on the top, and your initials written on the side) along with your application form into the coolers which are located in the bottom half of the -80C freezer. Select the size cooler most appropriate for your number of samples. Place the cooler containing the samples and application form into the upper half of the -80C freezer. For DNA samples, please use the insulated coolers found in the 4C refrigerator. Please do not write on the coolers, as the application form inside will provide us with your information. Samples that are left in any other type of box will NOT be transported to the core. Our courier will only deliver the blue cooler boxes, daily.

You will need a Dana-Farber ID card to enter the room. If you do not have one, please go to security in the lobby of the Dana building and request to be escorted to SM1129. The phone number for security is 617-632-3131 (or X 2-3131).

Note that this location is different from the Fish room on the bridge between the Smith Building and the Jimmy Fund Building.

GENE EXPRESSION PROFILING - 3' Arrays or Exon Arrays

Overview

A number of methods have been used to compare the absolute or relative expression of genes in two or more specimens. These include S.A.G.E. (Serial Analysis of Gene Expression), differential display, cDNA and oligonucleotide microarrays, and other methods. The Microarray Core offers a gene expression profiling service that uses oligonucleotide microarrays. Using this method, the RNA of interest is converted into cDNA, then amplified and labeled with biotinylated nucleotides by in vitro transcription to produce biotinylated cRNA. The cRNA is hybridized to a microarray chip. The bound cRNA is stained with fluorescent streptavidin. The microarray chip is then scanned using a laser, and the positions and intensities of the fluorescent emissions are captured. These measurements provide the basis of subsequent biostatistical analysis.

RNA

Amount (3' Array) - Investigators are asked to provide 15 µg of each RNA, if possible. If it is not possible to provide 15 µg of RNA, a smaller amount will often be suitable: for most specimens, 5 - 15 µg of RNA has been found to consistently produce excellent data. Results from analyses that begin with 1 - 4 µg of RNA are more likely to fail, but in our hands, approximately half of the specimens that begin with this amount produce excellent results. Ideally, the RNA should be in a volume of 10 µl. Some investigators have expressed an interest in analyzing very small amounts of RNA isolated from microdissected tissues or from subpopulations of cells purified by flow cytometry. At this time the Core does not offer a service to amplify the starting RNA before proceeding with the standard procedure.

Amount (Exon Array) - One microgram of total RNA in a volume of 3.2 µl is required.

Quality Control - Two quality control measures are carried out on a small aliquot of the submitted RNA samples: (1) a spectrophotometric analysis to confirm the concentration and to detect contaminating proteins and other molecules, and (2) a size fractionation procedure using a microfluidics instrument (Agilent Technologies) to determine whether the RNA is intact. An investigator will be notified if one or more of his/her samples are deemed unacceptable for analysis.

Microarray chips

Exon Arrays - All known and predicted exons are represented by four (>90% of exons) or less probes. Exon arrays are available for human, mouse and rat profiling. Please see the Pricing page of this site for kind of chips that are available and for their prices.

Gene Profiling Procedure

1. RNA is converted into cDNA using a T7 promoter-tailed oligo-dT primer in the synthesis of the first cDNA strand; second strand cDNA synthesis is then carried out.
2. The double-stranded cDNA is used as the template in an in vitro transcription (IVT) reaction catalyzed by T7 polymerase and containing biotinylated CTP and UTP in addition to the four unmodified ribonucleoside triphosphates.
3. The biotinylated complementary RNA (cRNA) is purified from the IVT reaction mixture using the RNeasy system (Qiagen). The cRNA is quantified spectrophotometrically and purity of the cRNA is also assessed by spectrophometric measurements. Those cRNA's that fall outside of an acceptable range will not be carried forward in the analysis. Should this occur, investigators would be notified and asked to provide a new RNA sample.
4. The purified cRNA is fragmented in order to facilitate the subsequent hybridization step.
5. The fragmented cRNA is added to a hybridization solution containing several biotinylated control oligonucleotides (for quality control), and hybridized to a microarray chip overnight at 45°C.
6. The chips are then transferred to a fluidics instrument that performs washes to remove cRNA that has not hybridized to its complementary oligonucleotide probe. The bound cRNA is then fluorescently labeled using phycoerythrin-conjugated streptavidin (SAPE); additional fluors are then added using biotinylated anti-streptavidin antibody and additional SAPE.
7. Each cRNA bound at its complementary oligonucleotide is excited using a confocal laser scanner, and the positions and intensities of the fluorescent emissions are captured. These measures provide the basis of subsequent biostatistical analysis. The data is transferred to a file-system from which it can be downloaded via a password-protected URL.

1. Control RNA's - the Core spikes in four in vitro synthesized, polyadenylated B. subtilis mRNA's into each submitted sample.
2. rRNA reduction - samples are enriched for mRNA by depletion of rRNA using rRNA capture probes and magnetic beads.
3. cDNA synthesis I - double-stranded cDNA is synthesized from the mRNA enriched sample starting with T7 promoter-tailed random hexamers.
4. cRNA synthesis - cRNA is synthesized in a T7 polymerase catalyzed reaction containing unmodified ribonucleoside triphosphates.
5. cDNA synthesis II - single-stranded cDNA is synthesized from the cRNA template using random primers and dUTP, dTTP, dATP, dCTP and dGTP.
6. cRNA hydrolysis - the cRNA is hydrolyzed by RNase H, and the single-stranded cDNA is purified.
7. Fragmentation of a single-stranded cDNA - Uracil DNA glycosylase and Apurine/Apyrimidinic Endonuclease I are used to fragment the cDNA at Uracil-containing positions.
8. Biotinylation - The fragmented cDNA is end-labeled with a biotinylated nucleotide analog.
9. The fragmented cDNA is added to a hybridization solution containing several biotinylated control oligonucleotides (for quality control), and hybridized to a microarray chip overnight at 45°C.
10. The chips are then transferred to a fluidics instrument that performs washes to remove cDNA that has not hybridized to its complementary oligonucleotide probe. The bound cDNA is then fluorescently labeled using phycoerythrin-conjugated streptavidin (SAPE); additional fluors are then added using biotinylated anti-streptavidin antibody and additional SAPE.
11. Each cDNA bound at its complementary oligonucleotide is excited using a confocal laser scanner, and the positions and intensities of the fluorescent emissions are captured. These measures provide the basis of subsequent biostatistical analysis. The data is transferred to a file-system from which it can be downloaded via a password-protected URL.

    SINGLE NUCLEOTIDE POLYMORPHISM (SNP) ANALYSIS

    The 500K SNP oligonucleotide microarray chip pair, which is comprised of a 250K StyI array and a 250K NspI array, is designed to discriminate alleles at approximately 500,000 dimorphic positions, in the human genome. These microarrays have been particularly useful in cancer research for carrying out genome-wide analysis of allelic changes (including deletions, amplifications, and gene conversions) in tumors. They are also useful in linkage analysis. At this time, only DNA from a fresh or frozen source can be analyzed on the SNP microarrays.

    DNA

    Isolation - The analysis in the Core begins with genomic DNA that is provided by an investigator. DNA extracted from either fresh or frozen tissue is suitable for analysis on the SNP arrays; At this point we are unaware of any particular DNA isolation protocol that produces DNA of a more suitable quality than another.

    Amount - Investigators are asked to provide 500 ng of genomic DNA (or 250 ng if a sample will be hybridized to only one of the 250K arrays), at a concentration of 50 ng/µl, from each specimen. If you are unable to obtain this amount, you may wish to try a procedure known as, "whole genome amplification", which has been shown to reliably amplify DNA by several orders of magnitude; the amplification does not alter the SNP determinations.

    SNP arrays

    The oligonucleotide arrays used in the Microarray Core are manufactured by Affymetrix. The manufacturing process involves the synthesis of ~24-40 different 25-mers per locus; the 25-mer probes are synthesized in situ using photolithography and combinatorial chemistry. Please see www.affymetrix.com/technology/manufacturing/index.affx for additional information on the manufacturing process.

    The ~24-40 probes are comprised of 3-5 groups of probes, each group of which is composed of (1) a perfect match for one of the allelic states (allele A); (2) a single base mismatch for allele A; (3) a perfect match for the other allelic state (allele B); and (4) a single base mismatch for allele B. The corresponding probes for each of the four complementary sequences are also part of the probe group. The position of the SNP within its component eight probes differs among the five groups.

    SNP Procedure

    1. The 500K analysis begins with 2 aliquots of 250 ng of DNA in each: one is cleaved with StyI; the other is cleaved with NspI.
    2. Linkers are ligated to the restricton fragments using T4 DNA ligase. The linkers provide a primer site for the subsequent PCR reaction.
    3. The fragments are amplified using Taq polymerase. The PCR products are purified from the primers and free nucleotides by ultrafiltration. They are then quantified spectrophotometrically, and are also assayed using size fractionation on a microfluidics device to determine whether the size distribution of products is as expected.
    4. The purified PCR products are fragmented by DNAse I in order to provide 3' hydroxyl groups for subsequent labeling, and to facilitate the subsequent hybridization step.
    5. The fragmented PCR products are labeled with a single biotin at each free 3'OH using terminal deoxynucleotidyl transferase and a dideoxy biotinylated nucleoside triphosphate.
    6. The biotinylated fragments are added to a hybridization solution containing a biotinylated control oligonucletide (for quality control), and hybridized to a microarray chip overnight at 49°C.
    7. The chips are then transferred to a fluidics instrument that performs washes to remove DNA that has not hybridized to its complementary oligonucleotide probe. The bound DNA is then fluorescently labeled using phycoerythrin-conjugated streptavidin (SAPE) followed by biotinylated anti-streptavidin, followed by SAPE.
    8. Each DNA bound at its complementary oligonucleotide is excited using a confocal laser scanner, and the positions and intensities of the fluorescent emissions are captured. These measures provide the basis of subsequent biostatistical analysis. The data is transferred to a database from which it can be downloaded via a password-protected URL.

    Tiling Arrays

    Human tiling arrays are designed by dividing the non-repetitive sequence of a genomic region or regions into contiguous 35 base pair units. A 25-mer is designed from each of these units (Promoter and Whole Genome arrays) or overlapping 7 base pair (ENCODE array) or 16 base pair (Chromosome 21/22 array) units The collection of these 25-mers comprises a tiling array. The arrays are useful for the localization of the genomic binding sites of transcription factors and other chromatin associated proteins. In addition to the human arrays (ENCODE, chromosome 21/22, promoter, whole genome), mouse arrays (promoter, whole genome), whole genome arrays for c.elegans, drosophila, s.cerevisiae, s.pombe and arabidopsis are also available.

    The process is as follows: an investigator performs chromatin immunoprecipitation, uses LM-PCR to amplify the DNA that has been extracted from the immunoprecipitate, fragments and biotinylates the PCR products, then submits those products to the Microarray Core for hybridization, staining and scanning.