title: DGRC-D. melanogaster-DGRC2-17328-v1
status: Public on Jun 20 2006
contact.city: Bloomington
contact.last: Drosophila Genomics Resource Center (DGRC)
contact.email: dgrc@cgb.indiana.edu
contact.state: IN
contact.address: 1001 E. 3rd St.
contact.department: Indiana University
contact.country: USA
contact.web_link: http://dgrc.cgb.indiana.edu/
contact.institute: Drosophila Genomics Resource Center (DGRC)
contact.phone: 812-855-5510
organism: Drosophila melanogaster
description: DGRC-2 transcriptome microarrays are spotted with gene-specific Drosophila melanogaster 70 N long oligonucleotides.  The oligos were designed against 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 65-69 nucleotides with a minimal Tm window, and locations biased towards the 3'-ends of transcripts.  Oligo Synthesis was completed by Illumina. The array also contains the following controls: a ubiquitously expressed gene (actin 5C), a spotting buffer-only control (150 mM NaPO4, pH 8.5), exogenous (non-Drosophila) probes: 9 from Arabidopsis, 2 from soybean, 1 from apple and Ambion’s  Array Control Sense Oligo Spots bacterial sequences that can be utilized with spiking RNAs, four exogenous markers - GAL4, lacZ, modified GFP, and FLPase, and a 70mer probe degradation long oligonucleotide set.  DGRC-2 arrays are printed using an Omnigrid 300 contact-printing instrument and 48 silicon split-pins on Corning UltraGAPS slides.
manufacture_protocol: Fabrication of DGRC-2 oligonucleotide microarrays
  Bogart, K., Cherbas, L., and Andrews, J.
  INTRODUCTION
  The Drosophila Genomics Resource Center (DGRC) provides the research community with access to genomics resources including microarrays. The first generation transcriptome microarrays fabricated by the DGRC (DGRC-1 arrays) are spotted with amplified DNA fragments. The DNA fragments are amplified from genomic DNA template by polymerase chain reaction using gene-specific primers. The primers were designed against release 1 of the D. melanogaster genome annotation, and the amplified DNAs were validated to perform well as microarray targets (Johnston et al. 2004). In recent annotations the coverage of the amplicons fell to 88% of the predicted genes (Costello and Andrews, 2006).
  Several recent studies have demonstrated the favorable performance of spotted oligonucleotide microarray platforms (Kane et al. 2000; Bozdech et al. 2003; Wang et al. 2003). Long oligonucleotides perform as well as cDNAs in detecting differential gene expression. Additionally, since oligonucleotides are shorter, they can be selected against unique sequences that distinguish between members of a gene family or alternative transcripts from a single gene. Motivated by the advantages of oligonucleotide microarrays and the need to update the existing Drosophila transcriptome platforms, the International Drosophila Array Consortium (INDAC) developed a set of oligonucleotides for microarray fabrication. This set of oligonucleotides is now being used by several Drosophila genomics facilities to fabricate transcriptome microarrays (FlyChip, CDMC, DGRC).
  Oligonucleotide design and synthesis
  The International Drosophila Array Consortium (INDAC) was formed to produce a widely available and uniform set of array reagents, so that microarray data collected from different studies may be more easily compared. On behalf of INDAC, the FlyChip group (Gos Micklem, Steve Russell, and their collaborators at the University of Cambridge) designed a set of long oligonucleotides to release 4.1 of the D. melanogaster genome, using a custom implementation of OligoArray2. Oligos were chosen to have minimal sequence similarity to other genes, size range between 69-65 nucleotides with a minimal Tm window, and location biased towards the 3'-end of transcripts. The final set of 15,158 oligonucleotides has the size distribution give in Table 1. The sequences of the oligonucleotides can be downloaded from DGRC platforms, and Flymine.
  
  Table 1. Distribution of oligonucleotide lengths
  Length (nt)	Total #	%
  69	9791	64.6
  68	749	4.9
  67	730	4.8
  66	713	4.7
  65	3175	20.9
  Annotation
  We have updated the annotation of the oligonucleotides to release v4.3 of the Drosophila genome (Rupali Patwardhan, James Costello, Justen Andrews, Peter Cherbas, unpublished). Full details can be found in the gene list master file which is posted for each DGRC-2 print run on the DGRC web-site; these files will be updated as new annotations of the Drosophila genome are released. Briefly, of the 15,158 oligonucleotides, 14,543 are designed against Drosophila sequences (the remainder being controls of various sorts); after correction for sequences that are repeated in the collection, there are 14,447 unique Drosophila oligo sequences. Of these, 14,047 are predicted to hybridize to transcripts defined in the v4.3 annotation of the Drosophila genome; 142 of them hybridize to transcripts from more than one gene. Altogether, of the 14486 genes in the v4.3 annotation of the Drosophila genome, 13548 (93.5%) are represented unambiguously in the INDAC collection of oligonucleotides. 72 of these
  genes express non-coding RNAs, some of which are not polyadenylated and therefore will not be detected with oligo-d(T)-primed fluorescent targets. In the INDAC set of oligonucleotides, no attempt was made to include probes specific for different transcripts  from individual genes; specific transcripts predicted to hybridize to each oligonucleotide probe are listed in the gene list master file on the DGRC web-site.
  The probe sequences were designed to be as close as possible to the 3’ end of transcripts. Probes generally hybridize within a few hundred nucleotides of the polyadenylation site of the transcripts against which they were designed, but some probes also hybridize to longer alternative transcripts. For each probe, we have calculated the distance from the 3’ end of the probe to the polyadenylation site of each predicted transcript to which it can hybridize; for all probe/transcript combinations, the mean distance is 723 N (S.D. = 576).
  Internal controls and array layout
  The set of INDAC oligonucleotides includes the following internal controls:
  1. Exogenous targets: 9 from Arabidopsis, 2 from soybean, 1 from apple. These serve as negative controls or targets for mRNA spike-in controls.
  2. Exogenous genes: GAL4, lacZ, modified GFP, and FLPase.
  3. A series of oligos designed to test for the intactness of RNA targets. The array contains multiple copies of a set of oligos made from different parts of the eukaryotic initiation factor (eIF-4a) transcript. Since most labeled targets are made as oligo(dT)-primed cDNAs, mRNA degradation and/or incomplete reverse transcription will result in overrepresentation of sequences from the 3’ portion of an mRNA. Therefore, a degraded RNA sample may be detected by a relative decrease in hybridization to the more 5’ eIF-4a probes. We have not yet experimentally validated this test.
  We have supplemented these controls with the following:
  1. The ubiquitously expressed genes actin 5c (act5c) and eiF-4a printed at a characteristic location in each of the 48 sub-arrays. These positive control spots serve as an easily recognized registration mark and as 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 negative control for background and check for carry-over between samples.
  3. Array Control Sense Oligo Spots (Ambion Inc.). These serve as additional negative controls and can also be utilized with spiking RNAs for normalization to verify the equivalence of labeling between channels.  Oligo Spots are PAGE-purified C6 amino-modified 70-mers single-stranded ("sense" orientation). Oligo Spot sequences have been designed for optimal annealing and minimal self-hybridization and are also checked for lack of cross hybridization to all sequences catalogued in public databases.
  The spotting buffer negative control, act5c and eiF-4a positive controls, and Array Control
  Sense spiking controls (Ambion) are printed by each of the 48 pins at the start of each block.
  This report gives an overview of the DGRC-2 oligonucleotide transcriptome microarrays
  printed by the DGRC using the INDAC set of oligos. It is intended to inform DGRC users
  about the platform and provide information on the production of the arrays.  The key issues in oligonucleotide microarray construction are 1)  the resuspension of lyophilized oligonucleotide in spotting buffer and 2) the deposition and immobilization of those DNAs onto glass slides. Below we describe the high throughput resuspension, printing and immobilization of oligonucleotides to create the DGRC-2 arrays.
  
  Part A. Resuspension
  Below, we will describe the Biomek FX methods for preparation of DNA for printing (deposition) by resuspending a 625 pmol plate of lyophilized oligonucleotides in 21 uL spotting buffer (i.e. giving a 30uM concentration) and aliquoting to 2 final 10 uL source print plates.  This method is repeated for each of the 41 384-well plates used for the DGRC-2 arrays.
  As a general cautionary note, it is important to avoid the use of latex gloves in all matters associated with the fabrication and use of microarrays. Latex contains a fluorescent component which will leach from the gloves and cause significant artifacts. Nitrile gloves are a readily available substitute and sh