GEO Logo
   NCBI > GEO > Accession DisplayHelp Not logged in | LoginHelp
GEO help: Mouse over screen elements for information.
Platform GPL284 Query DataSets for GPL284
Status Public on Jul 08, 2003
Title Human UniGene Set RZPD 1
Technology type spotted DNA/cDNA
Distribution non-commercial
Organism Homo sapiens
Description Preparation of high density cDNA arrays.

cDNA clone inserts (not sequence-verified) from eighty-eight 384-well microtitre plates from the Human Unigene 1 clone set (RZPD Deutsches Ressourcenzentrum für Genomforschung
and Boer et al. 2001) and a custom-made plate of an additional 258 clones (sequence-verified) were amplified by PCR in a 384-well format using M13 forward (5`-CGTTGTAAAACGACGGCCAGT-3´) and reverse (5´-TTTCACACAGGAAACAGCTATGAC-3´) primers. The PCR reaction was set up by dipping a 384-pin replicator (Genetix, UK) twice into the bacterial library plate and transferring the solution (approximately 0.2 µL) to a new PCR plate containing 25µL of PCR master mix in each well (final concentrations: 1 X PCR buffer (0.5M Tris-HCl (pH 8.6), 0.5M KCl, 15mM MgCl2, 1% Tween 20), betaine (1.5U), 0.2mM dNTPs, 0.2mM primers and 0.1U/µL Taq/Pfu). Amplification of PCR products was carried out as follows: 95ºC for 1 min, 94ºC for 15 sec, 59ºC for 15 sec, 65ºC for 55 sec, repeat of 59ºC for 15 sec, 65ºC for 55 sec for an additional 2 cycles, repeat of 94ºC for 15 sec, 59ºC for 15 sec, 65ºC for 55 sec for 29 cycles and finally 68ºC for 10 min. The sub-cycling step described improved product yield and stability. The quality of the PCR reaction was determined visually by slot-blot electrophoresis on 1% agarose gels.

Prior to spotting, PCR products were denatured by the addition of a final concentration of 0.1M NaOH to each well. The ~34,000 PCR products were repeatedly spotted 7 times in a 6 X 6 pattern (asymmetrically to ensure ease in determining filter orientation) on dry 23 X 23 cm Hybond N+ nylon membranes (Amersham Pharmacia) using a Genetix robot attached to a 384 pin gadget-head (250 mm pins). The 6 X 6 spotting blocks were organized into six fields of 384 spotting blocks (arranged 16 X 24), resulting in 2304 spotting blocks on each filter. Each 6 X 6 spotting block contained duplicate PCR products from 15 clones (14 clones in field 6), duplicate Arabidoposis thaliana clones (GenBank accession numbers AF104328 and U29785, derived from the Arabidopsis Biological Resource Center and DNA stock donor at Ohio State University) serve as guide spots during subsequent gridding steps, and four (six in field 6) empty positions to allow for an estimation of background intensity after hybridization. In order to determine the quality of the spotting, 1.58 µM fluorescin was added to the Arabidoposis thaliana control clone plate and the filter was scanned using an ‘in-house’ work station for high resolution fluorescence detection (MPI for Molecular Genetics, Berlin). In this way, unevenly or poorly spotted filters were discarded prior to hybridization. After spotting, cDNA on the filters was further denatured by floating filters (face up) in 0.4M NaOH for 2 min and neutralized with 5 X SSC (pH 7.5) for 2 min. The DNA was cross-linked to the filter by heat-baking at 80°C for 30 min and exposing to ultraviolet radiation (Stratalinker UV cross-linker (auto cross-link option); Stratagene). Filters were stored dry at 4°C until ready for use.

Boer, J.M., Huber, W.K., Sultmann, H., Wilmer, F., von Heydebreck, A., Haas, S., Korn, B., Gunawan, B., Vente, A., Fuzesi, L. et al. (2001) Identification and classification of differentially expressed genes in renal cell carcinoma by expression profiling on a global human 31,500- element cDNA array. Genome Res, 11, 1861-1870.

Web link
Contributor(s) Costello CM, Mauracher B, Waetzig G, Mah N, Eickhoff H, Schreiber S, Lehrach H
Submission date Apr 10, 2003
Last update date Jun 06, 2006
Contact name Nancy Mah
Phone +0049-431-597-3725
Organization name Christian-Albrechts-University Kiel
Street address
City Kiel
ZIP/Postal code D-24105
Country Germany
Samples (74) GSM5994, GSM5995, GSM5996, GSM5997, GSM5998, GSM26195 
Series (5)
GSE405 Microarray Comparison Using Normal Human Colonic Mucosa
GSE1510 Human UniGene Set RZPD 1 - Zipf's Normalization
GSE1710 Expression profiling in inflammatory bowel disease

Data table header descriptions
CLONE_ID IMAGE clone identifier
GB_LIST GenBank accession numbers;
UNIGENE UniGene cluster number
Gene_Name UniGene cluster name
3'_GB_ACC 3' GenBank accession number
5'_GB_ACC 5' GenBank accession number
reporter role

Data table
ID CLONE_ID GB_LIST UNIGENE Gene_Name 3'_GB_ACC 5'_GB_ACC reporter role x-Pos_GSE405 y-Pos_GSE405 x-Pos_GSE1710 y-Pos_GSE1710 SPOT_ID
01E01 IMAGE:60955 T40701,T39626 283619 zinc finger protein 236 T40701 T39626 measurement 167 138 69 287
02E01 IMAGE:76585 T50974,T51037 9250 ESTs T50974 T51037 measurement 71 282 69 285
03E01 IMAGE:22533 T74519 0 T74519 0 measurement 263 282 66 284
04E01 IMAGE:108297 T70562 337577 ESTs T70562 0 measurement 71 138 71 287
05E01 IMAGE:112590 T91141,T85660 0 T91141 T85660 measurement 263 138 71 286
06E01 IMAGE:115338 T87103,T87102 268615 ESTs T87103 T87102 measurement 167 282 68 284
07E01 IMAGE:121301 T96635 135265 Homo sapiens, clone MGC:10965 IMAGE:3633884, mRNA, complete cds T96635 0 measurement 167 139 67 282
08E01 IMAGE:124225 R02327,R02429 18026 ESTs R02327 R02429 measurement 71 283 71 282
09E01 IMAGE:127682 R09490,R09585 5943 rec R09490 R09585 measurement 263 283 66 287
10E01 IMAGE:25947 R11939 306189 DKFZP434F1735 protein 0 R11939 measurement 71 139 70 285
01E02 IMAGE:53191 T66743,T66744 165563 DnaJ (Hsp40) homolog, subfamily C, member 5 T66743 T66744 measurement 167 132 69 281
11E01 IMAGE:29138 R41285,R12891 11101 KIAA1232 protein R41285 R12891 measurement 263 139 69 286
12E01 IMAGE:31838 R41835,R17226 9315 HNOEL-iso protein R41835 R17226 measurement 167 283 66 282
13E01 IMAGE:34412 R44383,R24578 30957 Homo sapiens mRNA; cDNA DKFZp434E0626 (from clone DKFZp434E0626) R44383 R24578 measurement 167 143 67 284
14E01 IMAGE:36972 R49406 184786 TBP-interacting protein R49406 0 measurement 71 287 66 286
15E01 IMAGE:40297 R52083,R52177 93814 mitochondrial ribosomal protein L35 R52083 R52177 measurement 263 287 67 283
16E01 IMAGE:42689 R59910,R59909 177486 amyloid beta (A4) precursor protein (protease nexin-II, Alzheimer disease) R59910 R59909 measurement 71 143 165 287
17E01 IMAGE:132072 R26046,R23445 256583 interleukin enhancer binding factor 3, 90kD R26046 R23445 measurement 263 143 165 285
18E01 IMAGE:136536 R34362 0 R34362 0 measurement 167 287 162 284
19E01 IMAGE:140328 R66991 28107 ESTs R66991 0 measurement 162 141 167 287

Total number of rows: 34560

Table truncated, full table size 3579 Kbytes.

Download family Format
SOFT formatted family file(s) SOFTHelp
MINiML formatted family file(s) MINiMLHelp

Supplementary data files not provided

| NLM | NIH | GEO Help | Disclaimer | Accessibility |
NCBI Home NCBI Search NCBI SiteMap