Retired Platforms

The DGRC has discontinued distributing microarrays. This is in response to the fact that both Drosophila specific microarrays and custom microarrays are now readily available from commercial vendors. We are, however, continuing to provide support for arrays that have already been purchased. Gene lists and protocols are available on the Downloads page.

We are continuing our efforts to facilitate the availability and use of microarrays and other emerging genomics technologies. If you have any questions please feel free to contact us.

Drosophila melanogaster Gene Expression Microarray

The most comprehensive and up-to-date fruit fly expression array designed by the DGRC in collaboration with the Center for Genomics and Bioinformatics (CGB) at Indiana University and Roche NimbleGen Inc. The 12-plex microarray is designed from Dmel ver.5.25 gene builds from the finished D. melanogaster genome sequence assembly and contains 134,064 probes interrogating 64,020 exons and 17,511 distinguishable gene transcripts. The array is available from NimbleGen or the CGB, which offers full hybridization and data analysis service at an affordable price.

Advantages

Performance

The quality of gene expression data obtained from this custom designed NimbleGen microarray is demonstrated by dual-color target hybridization experiments of technical replicates of whole- body adult female RNA that is amplified, reverse transcribed, labeled by random priming with cy3 and cy5 fluorescent dyes and mixed onto arrays of the same 12x135K microarray chip. CGB protocols are published at: http://cgb.indiana.edu/publications/.

Drosophila melanogaster Expression Microarray and Service Specifications

Design name 100701_Dmel_JL_expr_HX12
Total number of probes 134,064 for each of 12 arrays on the same chip
Probe length 60mer
Target genes 64,020 exons from 14,518 genes, plus 17,511 gene transcripts
Annotation Each probe updated on each subsequent major annotation release
Data delivery Password protected project web page for viewing and downloading
Community service Published data archived for meta-analysis improving the genome database
Source for most current sequence and annotation Drosophila melanogaster dmel_r5.25_FB2010_02;
Annotation FlyBase R5.25;
ftp.flybase.net/genomes/Drosophila_melanogaster/dmel_r5.25_FB2010_02/




DGRC-2 Oligonucleotide Transcriptome Microarrays

DGRC-2 transcriptome microarrays are spotted with gene-specific Drosophila melanogaster 70 N long oligonucleotides, prepared in conjunction with INDAC.

The oligos were designed according to release 4.1 of the genome annotation using a custom implementation of OligoArray2. Oligos selected have minimal sequence similarity to other genes, size range between 69-65 nucleotides with a minimal Tm window, and locations biased towards the 3'-ends of transcripts.

Oligo Length

LengthTotal #%
69979164.6
687494.9
677304.8
667134.7
65317520.9

We have updated the annotation to release 4.3 in which the 15,158 oligonucleotides correspond to roughly 93% of the annotated genes. Oligo Synthesis was completed by Illumina. We have supplemented the exon-specific oligonucleotides with the following controls:

DGRC-2 arrays are printed using an Omnigrid 300 contact-printing instrument and 48 silicon split-pins on Corning UltraGAPS slides. Probe DNA is dissolved in 150 mM NaPO4, pH 8.5.

DGRC-1 Amplicon Transcriptome Microarrays

DGRC-1 amplicon transcriptome microarrays are spotted with DNA fragments amplified from genomic DNA using gene-specific oligonucleotide primer pairs. The primers were made by Incyte Genomics and were generously donated to the DGRC by Incyte Genomics and Brian Oliver (NIDDK, National Institutes of Health).

The primers were designed against release 1 of the annotated genome sequence and were selected to amplify fragments which have minimal sequence similarity to other genes, size range between 100-600 bp, and location biased towards the 3'-end of transcripts. The 14,151 primer pairs correspond to 94% of the release-1 predicted genes and roughly 88% of the version 4.3 annotated genes. We have supplemented the Incyte Genomics exon-specific primers with the following controls:

  1. An ubiquitously expressed gene (actin 5C) printed at a characteristic location in each of the 48 sub-arrays. This serves as an easily recognized registration mark and also serves a check on the uniformity of hybridization across the slide.
  2. A spotting buffer-only control printed in each of the 48 sub-arrays. This serves as a control for background and check for carry-over between samples.
  3. 15 exogenous(non-Drosophila) probes: 7 from Arabidopsis and 8 from bacteria.
  4. Array Control PCR Spots (Ambion Inc.). These serve as additional negative controls and can also be utilized with spiking RNAs.
  5. The exogenous markers - GAL4, lacZ, and FLP.

The DGRC arrays are printed using an Omnigrid 300 contact-printing instrument and 48 silicon split-pins on Corning GAPS II slides. Probe DNA is dissolved in 3XSSC plus 1.5M Betaine.

Choosing Between DGRC-1 and DGRC-2

General: DGRC-1 has proven a robust platform and a priori we anticipate that it will continue to be usefull for those using pormymorphism rich RNA samples. It also may be more convenient for those using some RNA amplification protocols (see below). On the other hand DGRC-2 represents an up-to-date annotation of the genome, our tests reveal several specific instances in which it gives more accurate results, and we find that it gives approximately equivalent or better signal/noise.

RNA Amplification: Users planning to amplify their RNA samples to prepare microarray probes should be aware that the use of oligo arrays may be incompatible with some labeling protocols. The oligos spotted on DGRC-2 arrays are identical to the DNA coding stand (complementary to the template strand) and therefore complementary to cDNA that is reverse transcribed from mRNA. Users intending to amplify RNA samples should be aware that RNA amplified using the Eberwine procedure is the complement to the starting mRNA. This amplified RNA can, itself, be labeled and is then suitable for hybridization to oligonucleotide microarrays. Protocols that call for amplified RNA to be labeled through a reverse transcription reaction will generate labeled cDNA that is identical to the DNA coding strand and thus not suitable for hybridization. We have successfully tested the Ambion Amino Allyl MessageAmp II aRNA Amplification Kit, which yields amino allyl labeled RNA that is the complementary strand to the starting mRNA, and is suitable for hybridization to oligonucleotide microarrays.

Experimental comparison of DGRC-1 and DGRC-2: For one experimental comparison, go here.

Genome Tiling Path Microarrays

The DGRC is distributing Genome Tiling Path microarrays. These microarrays are spotted with DNA fragments amplified from genomic DNA using sequence-specific oligonucleotide primer pairs. The partially overlapping DNA fragments form a "tiling" coverage of the entire euchromatic genome of Drosophila melanogaster. This platform was developed by Kevin White's group (Kevin White and Tong-Rue Li, University of Chicago/Yale University) and the UK FlyChip group (Steve Russell and Gos Micklem, Cambridge University).

The probes are being prepared by the White group by PCR amplification of genomic DNA template using sequence-specific oligonucleotide primer pairs. The primers were designed based on the version 4.2 of the annotated genome sequence (except for a small number of pilot primers that were based on version 3.2); each primer recognizes 22 bp of unique genome sequence. The PCR fragments are between 900 and 1,300 bp in length (mean 1,100, median 1,100, SD 124 bp). DNA for the PCR is prepared from the strain that was used for the genome Drosophila melanogaster sequencing step (y1; cn1 bw1 sp1).

The microarrays are printed using an Omnigrid (GeneMachines) contact-printing instrument and stainless steel pins (TeleChem) on poly-lysine glass slides. Probe DNA was dissolved in 3XSSC, 0.005% sarcosyl at a median concentration of 0.6 ug/ul for printing.

Test Microarrays

The test arrays are intended for pilot experiments to optimize experimental conditions and are identical to the full transcriptome microarrays except that they contain fewer spots. The test arrays are produced using the same protocols as the comprehensive amplicon arrays so that experimental conditions optimized using the test arrays will translate directly to the full amplicon arrays.

Test Array
This image is a sample image to demonstrate array layout. Quality Control images from individual print runs are found under Downloads.

DGRC-1-test arrays contain 196 spots and include 24 Drosophila genes and the same controls spotted on full transcriptome arrays (Act5C, spotting buffer only, exogenous targets serving as negative controls or spiking controls, and the exogenous markers GAL4, lacZ and FLP).

DGRC-2-test arrays include 89 Drosophila genes and the controls: Act5C, spotting buffer only, 6 Arabidopsis exogenous targets, and 8 bacterial exogenous targets serving as negative controls or spiking controls.

Versioning

For each batch of microarrays we will provide an up-to-date gene list and quality control data. We will also archive these data and make them available to download from this website.

Quality control

We conduct quality control tests on each PCR amplified DNA sample and selected slides from each batch (print run) of microarrays. These data are recorded and tracked in a Laboratory Information Management System (Nautilus Thermo Labsystems) and will be supplied to users and made available for downloading from this website.

One slide from each print run is hybridized with an oligonucleotide complementary to a barcode element present at three unique locations in every 96 well plate of primers. This test confirms that all the sample plates were in the correct order and orientation.

Barcode Element

A slide from each print run is stained for DNA content with a random labeled oligdeoxynucleotide (nonamer) and the fluorescence intensity of each spot is recorded. Any missing spots or systematic defects in the printing are flagged.

Nonomer test

Two slides from each print run are hybridized with a complex target - labeled cDNA from whole adult males and whole adult females. Signal intensities and background intensities are compared to those from previous batches.

labeled cDNA from whole adult males and whole
      adult females

Each Test Array print run is subjected to the same Quality control tests as a Full-Transcriptome Array with the exception of the Barcode Element Hybridization, because the Barcode Element is not printed on the Test Arrays.


Citing the DGRC

When publishing experiments using materials obtained from the DGRC please cite the Drosophila Genomics Resource Center, supported by NIH grant 2P40OD010949, in the acknowledgments. Your cooperation helps us when we need to renew our grant.