生物芯片实验手册（NIH）

OVERVIEW

Fluorescent cDNA microarray technology is useful for making estimates of the abundance of particular messages relative to a designated source of mRNA that serves as a reference point. Commercial support of this technology has recently reached a level where it is reasonable for departments or large laboratories to consider setting up their own cDNA array facility. This set of protocols is intended to serve as a basic introduction to making and using cDNA microarrays for those embarking on this path. There are three fundamental types of operations required in a cDNA microarray experiment. The first operation, BASIC PROTOCOL 1, cDNA AMPLIFICATION AND PRINTING, deals with making the cDNA microarray itself. It is necessary to collect an inventory of cDNA bacterial clones that represent the genes whose message abundance you wish to survey. Plasmid templates are made from these clones and used as PCR substrates to produce DNA representations of the EST inserts. The PCR products are then purified and spotted onto poly-L-lysine coated microscope slides. In the second operation, BASIC PROTOCOL 2, RNA EXTRACTION AND LABELING, RNA is extracted from the cell samples to be examined, purified, and used as the substrate for reverse transcription in the presence of fluor-derivatized nucleotides. This procedure provides the tagged representations of the mRNA pools of the samples that will be hybridized to the gene-specific cDNA detectors immobilized on the microarray. The third fundamental operation, BASIC PROTOCOL 3, HYBRIDIZATION AND DATA EXTRACTION, covers the steps in which fluor-labeled cDNAs hybridize to their complements on the microarray, and the resulting localized concentrations of fluorescent molecules are detected and quantitated.

FABRICATION

This protocol describes the steps required to produce a cDNA microarray. Gene-specific DNA is produced by PCR amplification of purified template plasmid DNAs from cloned ESTs. The PCR product is purified by ethanol precipitation, thoroughly resuspended in 3XSSC, and printed onto a poly-L-lysine coated slide.

Materials, Reagents & Solutions

96 well alkaline lysis miniprep kit (Edge BioSystems, Gaithersburg, MD)
LB Broth (Biofluids, Rockville, MD)
Superbroth (Biofluids, Rockville, MD)
.dATP, dCTP, dGTP, dTTP, 100 mM each #27-2035-02, store frozen, -20°C (Pharmacia, Peapack, NJ)
PCR primer AEK M13F (5'-GTTGTAAAACGACGGCCAGTG-3') and AEK M13R (5'-CACACAGGAAACAGCTATG-3') at 1 mM concentration, store frozen, -20°C
10X PCR Buffer, # N808-0189, and Ampli-Taq DNA polymerase, # N808-4015 store frozen, -20°C (Perkin Elmer, Norwalk, CT)
Carbenicillin (Gibco-BRL, Rockville, MD)
Ethanol (200 Proof USP Ethyl Alcohol)
1M Tris-HCl (pH 8)
0.5M NaEDTA (pH 8)
T Low E Buffer
20X SSC
Glycerol (enzyme grade)
Sodium Acetate (tri-hydrate)
Boric Acid
Sodium Hydroxide (1M)
Glacial Acetic Acid
Succinic anhydride, #23969-0 and 1-methyl-2-pyrrolidinone, # 32863-4 (Aldrich Chemical Co., St. Louis, MO)
Diethyl Pyrocarbonate (DEPC) treated H2O
Master set of clone-purified, sequence verified human ESTs (e.g. gf211 release, Research Genetics, Huntsville, AL)
96 pin inoculating block (#VP 4088, V&P Scientific, Inc, San Diego, CA)
Airpore Tape Sheets, (# 19571, QIAGEN Inc., Valencia, CA)
Sterile 96-well plate seals, (e.g. # SEAL-THN-STR (Elkay Products, Inc., Shrewsbury, MA)
96-well U-Bottom Microtiter Plates, #3799 and 96-well V-Bottom Microtitre Plates, #3894 (Corning Inc., Corning, NY)
Thin wall PCR plate and Cylcleseal PCR plate sealer (e.g. #1038-50-0 and #1044-39-4, Robbins Scientific Corp. Sunnyvale, CA)
household one-gallon sealable storage bags (e.g. Glad Lock)
heat sealable storage bags and heat sealer
0.2mm Sterile Filtration unit
Diamond scribe for writing on slides
Pyrex baking dish (~ 24x34x5 cm)
UV transparent plastic wrap (e.g. Glad Cling Wrap)
30 slide rack (stainless steel) #113 and 30 slide glass tank, #122 (Shandon Lipshaw, Pittsburgh, PA)
1L glass tank
1L glass beaker
1L graduated cylinder
Stir bar
Slide Box (plastic with no paper or cork liners), (e.g. #60-6306-02, PGC Scientific, Gaithersburg, MD)
PCR heat cycler (e.g. DNA Engine Tetrad, MJ Research, Waltham, MA)
Centrifuge with a horizontal ("swinging bucket") rotor with a depth capacity of 6.2 cm for spinning microtiter plates and filtration plates (e.g. Sorvall Super T 21, Sorvall Inc., Newtown, CT)
37°C Shaker incubator with holders for deep-well plates
37°C Waterbath
65°C Incubator
Vortex mixer
Immunowash microtiter plate washer, #1575 (BioRad, Hercules, CA)
pH Meter
Platform Shaker
UV Stratalinker 2400, (Stratagene La Jolla, CA)
Stirrer/Hotplate
Robotic slide printer
- 80°C Freezer
- 20°C Freezer