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Amplification of Mouse cDNAs for Microarrays Using the Eppendorf ,,, MasterTaq Kit

Katrine Verdun, Richard Rouse, and Gary Hardiman
BIOGEM, Division of Biology, University of California San Diego, La Jolla, CA 92093-0349
e-mail: ghardiman@ucsd.edu
www.biogem.ucsd.edu

Introduction

Microarray technology, primarily advanced by Pat Brown and his colleagues at Stanford University, makes use of a robotic spotting device or "microarrayer" to spot DNA sequences onto "derivatized" glass slides (1). These arrays are then hybridized with cDNA probes and analyzed using methods conceptually similar to those described for Affymetrix gene chips (2). Since the cost of producing an individual array is relatively small, spotted arrays are highly versatile and can make use of a wide variety of clone sets. Microarrays thus enable individual investigators to perform large-scale analysis of model organisms or to customize arrays for special genome applications.

The cDNAs spotted on microarrays are typically PCR products, amplified by PCR from plasmid miniprep DNA of cDNA clones. Amplification directly from clones in culture has been recently reported (3). This is more advantageous in high-throughput applications in that it is more cost efficient, considerably less labor intensive, and minimizes sample cross-contamination. The recommended concentration for spotting on aminosilane and polylysine slides is 200400 fmol/l. Lower concentrations than this will result in poor signals, and higher concentrations are not recommended as target spots are seen to smear. Microarray hybridizations work poorly with weak PCR products. Consequently, PCR amplification is optimized to maximize DNA yield. In this report we describe the use of the Eppendorf MasterTaq Kit in the preparation of targets for a mouse ~8 K microarray.

Materials and Methods

PCR amplification
Selected clones were inoculated into 96-well Falcon U-bottom plate (Nunc) containing LB/Ampicillin (50 mg/ml) and were placed in a shaking incubator (200 rpm) overnight at 37C. PCR reactions (50 l) were assembled using a Qiagen BioRobot 3000 and consisted of 10 M dNTP mix, 20 M primer mix (forward primer 5'-CTG CAA GGC GAT TAA GTT GGG TAA C-3' and reverse primer 5'-GTG AGC GGA TAA CAA TTT CAC ACA GGA AAC AGC-3') and 1 l of overnight culture. Amplifications were carried out using 7.5 Units of polymerase in the recommended buffer, in accordance with the instructions provided by the manufacturer. Thermocycling was performed on an MJ Research Tetrad. Cycling conditions were as follows: an initial five-minute denaturation at 95C to lyse the cells and release the plasmid DNA, 35 cycles (94C for 1 minute, 58C for 1 minute, 7 2C for 4 minutes), followed by 10 minutes at 72C. Conditions recommended by manufacturers of Hot Start polymerases typically include a 15-minute rather than a 5-minute initial denaturation step. One-fifth of the PCR amplification was analyzed on a 0.8% agarose gel containing ethidium bromide and was visualized with an AlphaImager 2200 Documentation and Analysis system.

The mouse GEM 1 Clone List (build 35) containing 8,734 clones was purchased from Incyte Pharmaceuticals (Palo Alto, CA). For amplification of this mouse Unigene set, reactions were carried out in a 96-well plate format using 3 l of overnight culture and either 7.5 or 3.75 U MasterTaq respectively, in a final volume of 150 l. Successful PCR amplifications were verified by electrophoresis on agarose gels. PCR products were purified on a BioRobot 3000 (QIAGEN, Santa Clarita, CA) using the QIAquick 96 PCR BioRobot Kit. The yield of PCR amplicon was assayed using the Picogreen dsDNA quantitation kit (Molecular Probes) and a Bio-Tek FLX800 microplate fluorescence reader.

Preparation of fluorescently labeled cDNA
One g of polyA+ RNA (mouse spleen and liver, respectively) were converted into fluorescently labeled cDNA by incubation with an oligo dT primer (44 M final) at 70C for 10 minutes in a final volume of 9 l. The solution was then mixed with 11 l of a solution 1.8X first strand buffer, containing 18 mM DTT, 900 M dATP, 900 M dGTP, 900 M dTTP, 230 M dCTP, 230 M Cy3- or Cy5-dCTP (APBiotech, UK), 20 U of RNasin (Promega), and 200 U Superscript II reverse transcriptase (Life Technologies). The reaction was incubated at 42C for 2 hours and heated at 100C for 5 minutes. RNA was hydrolyzed by the addition of 1 l of 10 N NaOH and subsequent incubation at 37C for 15 minutes. The reaction was neutralized by the addition of 5 l of 1M Tris pH 7.2 and 2 l of 5N HCl, and cDNA probes were purified using a QIAquick PCR purification kit (Qiagen, Santa Clarita, CA). Typically, one-third of each reaction was used per hybridization.

Microarray fabrication, hybridization, washing, scanning, and data analysis
PCR products (1,152 in total) derived from the mouse Unigene set and control cDNA clones were printed on a reflective slide (APBiotech, UK). DNA clones were heat denatured for 3 minutes at 95C, chilled on ice, and arrayed using a Molecular Dynamics (Sunnyvale, CA) Generation III spotter. After printing, the microarrays were allowed to dry completely at ambient conditions. The slides were pretreated with 2x SSPE, 0.2% SDS at 55C. The cDNA probe was lyophilized and redissolved in ~32 l Microarray Hybridization Buffer Version 2 (APBiotech, UK). The solution was added to the microarray and a coverslip was applied. Hybridization was allowed to proceed for 1418 hours at 42C. The microarray was washed with 1X SSC/0.2% SDS for 5 minutes at 45C followed by two 5-minute washes with 0.1X SSC/0.2% SDS at room temperature. The microarray was rinsed briefly with water, dried with nitrogen, and scanned using a Molecular Dynamics Generation III scanner.

Results and Discussions

For the analysis of gene expression in eukaryotes, expressed sequence tag (EST) data represent an invaluable resource for gene identification. EST's are single pass partial cDNA sequences. In general, cDNA clones are selected to represent as many unique transcripts as possible. One such method to group transcripts is the Unigene clustering of data sets which attempts to identify unique human transcripts within EST data (http://www.ncbi.nlm.nih.gov/UniGene). In amplifying EST collections for the production of microarrays, the yield of each PCR amplicon is a very important consideration. The optimal concentration of DNA for successful microarray experimentation is typically 200 g/ml. Hybridizations using non-covalent attachment chemistries (e.g. polylysine, aminosilane) generally work poorly with DNA spotted at concentrations lower than this. When amplifying thousands of clones, the main objective is to minimize the total number of reactions needed to generate sufficient material for printing. A high yield of PCR product includes plentiful amounts of DNA available for spotting, a reduction in cost and labor, and ultimately the production of greater numbers of arrays per PCR reaction.

Comparison of the efficiency of amplification of thermostable polymerases from bacterial cultures
Eppendorf MasterTaq and ten other polymerases were evaluated for the ability to amplify directly from overnight bacterial cultures. Approximately 1 l of the overnight culture was used to evaluate each enzyme in a reaction volume of 50 l. The main criteria for evaluation of the polymerases were specific amplification and maximum yield of the desired PCR product (Fig. 1).

Figure 1
The Eppendorf MasterTaq (#2) and ten other polymerases were evaluated for the ability to amplify directly from overnight bacterial cultures.

Polymerases:

1. Eppendorf Taq
2. Eppendorf MasterTaq
3. Competitor A
4. Competitor B
5. Home Brew
6. Competitor C
7. Competitor D
8. Competitor E
9. Competitor F
10. Competitor G
11. Competitor H


Upon comparison of ten enzymes, it was observed that enzymes 5 and 6 failed to generate specific products. Enzymes 1, 2, 3, 4, and 7 all performed well in terms of yield of PCR product and absence of non-specific amplicons, but enzyme 2 had no failures. The use of enzymes 8, 9, 10, and 11 all resulted in the amplification of extraneous bands. Given that enzyme 2 (Eppendorf MasterTaq) amplified all targets specifically, it was then investigated as to whether or not a dilution of the enzyme would still result in successful amplification from an overnight culture. The enzyme was tested at full strength (7.5 U/150 l reaction) and at half strength (3.25 U/150 l reaction). We observed that a one-half strength dilution of the enzyme yielded successful amplification of DNA from bacterial clones (Fig. 2).


Figure 2
Comparison of Eppendorf MasterTaq at half-strength and full-strength concentrations in amplifying 96-well plate overnight cultures of mouse clones.

Amplification of the mouse unigene set using the Eppendorf MasterTaq Kit

The Eppendorf MasterTaq Kit was used to amplify all ~8 K targets that comprise the mouse Unigene set. In total 8,734 amplifications were performed. Figure 2 shows agarose gel analyses of representative mouse cDNA amplifications using the Eppendorf MasterTaq kit in a 96-well plate format. In general, strong unique amplicons were observed. The results were classified as follows:

~ 7,997 single amplicons (strong bands)
~ 359 single amplicons (weak bands)
~ 293 multiple bands (typically two or more)
~ 85 failures

The cDNA clones that generated multiple bands were re-racked and amplified with a nested primer set (data not shown). For the most part, multiple bands were still observed indicating that these cDNAs represent mixed clones. This signifies ~3.4% contamination, typically found in amplification of a large clone set.

Amplicon Yield Per 150 l Reaction

Source Plate Name Avg. Total ng/Plate 4W-6Z 5541.5 7W-9Z 5367.9 10W-12Z 5174.5 13W-15Z 4848.7 16W-18Z 5187.3 19W-21Z 4169.5 22W-6YAmm. 4401.6 Avg Total ng in Mouse EST 4955.9 Table 1
Yield of DNA from amplification of the mouse Unigene set. Eight samples from 96-well plates containing purified DNA were assayed for DNA concentration. The plates assayed were numbered 4W, 4X, 4Y, 4Z, 5W, etc. through 22Z. There were five additional plates called 24W, 24X, 24Z, 23W, and 6Yamm respectively.

Quantitation of amplicon yield

All of the PCR products were purified on a BioRobot 3000 (QIAGEN, Santa Clarita, CA) using the QIAquick 96 PCR BioRobot Kit. Representative DNA samples from the entire set of amplifications were then quantitated using the Picogreen kit (Molecular Probes) to determine an estimate of the yield of purified PCR product. The results are shown in Table 1. The mean clone total yield was 4955.9 ng. The DNAs were resuspended at a concentration of ~200 g/ml in 25 l 50% DMSO-H2O. Many microarray spotters use 200 nl per print run and have a void volume of ~5 l. Assuming the spotter can print 36 arrays per run, this equates in theory to approximately 3,600 microarray slides. The working number of slides however is lower than this due to sample evaporation and other printing variables.

Screening of a mouse microarray with cDNA probes generated from mouse liver and spleen polyA+ RNAs

The final experiment performed was to verify that the PCR products amplified using the Eppendorf MasterTaq Kit generated strong specific signals in a microarray experiment. We printed 1,152 PCR products representing mouse cDNA clones and appropriate controls on a reflective slide (APBiotech, UK). Fluorescent probes were synthesized from mouse spleen and polyA+ RNAs and were used to screen the microarray (Fig. 3). Green, red, and yellow spots were observed, indicating positive hybridization of the labeled probes to the printed PCR products. The enlarged area shows the signal color range observed when using cyanine probes, Cye3 spleen, and Cye5 liver. Green (Cye3) and red (Cye5) spots indicate differential expression of these particular mRNAs sequences in spleen and liver respectively, whereas yellow spots indicate similar levels of expression in both tissues.

1152 cDNA clones derived from the mouse uniqene set and controls were amplified with the Eppendorf MasterTaq Kit and printed on derivatized glass slides. Fluorescent probes were synthesized from spleen and polyA+ RNAs and used to screen the microarray. The enlarged areas shows signal color range when using cyanine probes, Cye3 spleen and Cye5 liver. Green, red, and yellow spots indicate positive hybridization from the labeled probes. Green (Cye3) and red (Cye5) spots indicate that the targets are expressed differentially. Figure 3
The 1,152 cDNA clones derived from the mouse Unigene set and controls were amplified with the Eppendorf MasterTaq Kit and printed on derivatized glass slides. Fluorescent probes were synthesized from spleen and polyA+ RNAs and were used to screen the microarray.

The enlarged area shows signal color range when using cyanine probes Cye3 spleen and Cye5 liver. Green, red, and yellow spots indicate positive hybridization from the labeled probes. Green (Cye3) and red (Cye5) spots indicate that the targets are expressed differentially.

Conclusion

For generation of PCR amplicons for microarray analyses, the Taq from the Eppendorf MasterTaq kit performs exceptionally well, even when used at one-half of the recommended concentration. High yields of specific amplicons are obtained which can be used to generate high quality microarrays.

References

(1) Schena, M.; Shalon, D.; Davis, R.W.; and Brown, P.O. (1995). Quantitative monitoring of gene expression patterns with complementary DNA microarray. Science 270: 467-470.

(2) Lockhart, D.J.; Dong, H.; Byrne, M.C.; Follettie, M.T.; Gallo, M.V.; Chee, M.S.; Mittmann, M.; Wang, C.; Kobayashi, M.; Horton, H.; and Brown, E.L. (1996). Expression monitoring by hybridization to high-density oligonucleotide arrays. Nature Biotechnol. 14: 1675-1680.

(3) Hegde, P.; Rong Qi; Abernathy, K.; Gay, C.; Dharap, S.; Gaspard, R.; Earle-Hughes, J.; Snesrud, E.; Lee, N.; and Quackenbush, J. (2000). A Concise Guide to cDNA Microarray Analysis. Biotechniques 29: 548-562.









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