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Platform GPL10 Query DataSets for GPL10
Status Public on May 31, 2001
Title Human Unigene I, part 2
Technology type spotted DNA/cDNA
Distribution non-commercial
Organism Homo sapiens
 
Description Human cDNA filter from the Resource Centre of the German Human Genome Project. Each of the two parts (GPL9 and GPL10) contains half of 31,488 PCR amplificates of human cDNAs selected as representatives of UniGene clusters (Build #17, NCBI).

Selection of a Global Human Clone Set:

In order to generate a non-redundant human clone set, we postprocessed the UniGene clusters (Build 17, NCBI), which represent a large number of human genes. All processing steps are part of the GeneNest software (Haas et al., 2000), that additionally provides an interactive graphical interface to the postprocessed UniGene database (http://www.dkfz.de/tbi/services/GeneNest/index). To identify the most reliable, representative clone from each cluster, we analyzed the following criteria (sorted according to their importance): (1) Availability of clones at the RZPD. (2) Quality of cDNA library of origin. (3) Presence of more than one read of the same clone in a cluster, ensuring a higher confidence in the sequence - clone relationship. (4) Calculated insert size, selecting for larger inserts. (5) Presence of a polyA signal. We used a fuzzy logic based rule system to combine all clone selection criteria in order to obtain a quality measure of each clone in the entire UniGene set. For each cluster we selected the clone with the highest quality as representative. To estimate the redundancy of the global clone set, we re-sequenced over 2,700 clones of the set and found 12.8% wrongly assigned clones. All of these belonged to clusters that were already represented by another clone. Therefore, the overall redundancy of the clone set was estimated to be 1.44-fold, and the 31,500 clones on the cDNA array represent an estimated 21,875 different transcripts.

31,500 Clone Human cDNA Array:

From the Human UniGene 1 clone set, cDNA inserts of the clones from the 82 first 384-well microtiter plates were amplified by PCR in a 384-well format (MJ Research, Boston, Massachusetts) using M13 forward (5'-CGTTGTAAAACGACGGCCAGT-3') and reverse primers (5'-TTTCACACAGGAAACAGCTATGAC-3'). The 31,488 PCR products were transferred in a 4x4 pattern onto a set of two 22x22 cm Hybond N+ nylon membranes (Amersham Pharmacia Biotech, Uppsala, Sweden) soaked in 0.4 M NaOH using a Picking-Spotting-Robot (Linear Drives LDT, Essex, UK) with 400 ┬Ám pins (Genetix, Hampshire, UK). After spotting the arrays were carefully floated for 2 minutes on 0.4 M NaOH and 5 x SSC (pH 7.5) successively, air dried and cross-linked by UV. Every 4x4 block contained one spot with PCR product from the bacterial kanamycin resistance gene to serve as a guide spot in automated image analysis, one empty spot to serve as background measurement and DNA from seven clones spotted in duplicate. On each filter, a control plate containing putative housekeeping genes and positive and negative controls was spotted. To assure even quality between subsequent rounds of hybridization, cDNA arrays were pre-stripped before their first use (Hauser et al., 1998). To check for filter quality, M13 forward oligonucleotide hybridizations were carried out.
Keywords = membrane, filter
 
Contributor(s) Huber W, Boer J, Sueltmann H, Wilmer F, Heydebrec A, Haas S, Korn B, Vente A, Vingron M, Poustka A
Citation(s) 12199289
Submission date May 30, 2001
Last update date Oct 28, 2005
Contact name Wolfgang Huber
E-mail(s) w.huber@dkfz.de
Phone +49-6221-424709
Fax +49-6221-423470
URL http://www.dkfz-heidelberg.de/abt0840
Organization name German Cancer Research Center
Department Molecular Genome Analysis
Street address Im Neuenheimer Feld 280
City Heidelberg
ZIP/Postal code 69120
Country Germany
 
Samples (218) GSM181, GSM182, GSM183, GSM184, GSM185, GSM186 
Series (2)
GSE3 Renal Cell Carcinoma Differential Expression
GSE112 Distinctive gene expression patterns in gastrointestinal stromal tumors identified with cDNA arrays

Data table header descriptions
ID
X X coordinate
Y Y coordinate
CLONE_ID Visit http://www.rzpd.de
SPOT_ID Spot identifier
RZPD_ID Visit http://www.rzpd.de
GB_LIST
DESCRIPTION description

Data table
ID X Y CLONE_ID SPOT_ID RZPD_ID GB_LIST DESCRIPTION
1 1 1 IMAGE:767062 IMAGp950P2486 aa424310, aa451748
2 2 1 IMAGE:277995 IMAGp950P2444 n63451, n94732
3 3 1 IMAGE:297869 IMAGp950P2450 n70041 h.sapiens mrna for tgif protein,h.sapiens mrna for tgif protein,tgif
4 4 1 IMAGE:339113 IMAGp950P2456 w52973, w60453 homo sapiens ser/arg-related nuclear matrix protein (srm160) mrna, complete cds,homo sapiens ser/arg-related nuclear matrix protein (srm160) mrna, complete cds,srm160
5 1 2 IMAGE:366436 IMAGp950P2462 aa026378, aa026388
6 2 2 heterologous
7 3 2 IMAGE:727916 IMAGp950P2480 aa393468, aa435510 ests, weakly similar to ding [h.sapiens]
8 4 2 IMAGE:489566 IMAGp950P2468 aa098885, aa098895 homo sapiens advillin mrna, complete cds,avil,ests, highly similar to villin [gallus gallus]
9 1 3 IMAGE:489566 IMAGp950P2468 aa098885, aa098895 homo sapiens advillin mrna, complete cds,avil,ests, highly similar to villin [gallus gallus]
10 2 3 IMAGE:297869 IMAGp950P2450 n70041 h.sapiens mrna for tgif protein,h.sapiens mrna for tgif protein,tgif
11 3 3 IMAGE:366436 IMAGp950P2462 aa026378, aa026388
12 4 3 IMAGE:767062 IMAGp950P2486 aa424310, aa451748
13 1 4 IMAGE:727916 IMAGp950P2480 aa393468, aa435510 ests, weakly similar to ding [h.sapiens]
14 2 4 IMAGE:277995 IMAGp950P2444 n63451, n94732
15 3 4 IMAGE:339113 IMAGp950P2456 w52973, w60453 homo sapiens ser/arg-related nuclear matrix protein (srm160) mrna, complete cds,homo sapiens ser/arg-related nuclear matrix protein (srm160) mrna, complete cds,srm160
16 4 4 empty
17 5 1 IMAGE:759522 IMAGp950P2386 aa442734
18 6 1 IMAGE:277420 IMAGp950P2344 n34517, n47730 homo sapiens integrin alpha-7 mrna, complete cds,integrin, alpha 7b : itga7,itga7
19 7 1 IMAGE:296799 IMAGp950P2350 n74087, w01173
20 8 1 IMAGE:328211 IMAGp950P2356 w31901, w39264 human chromosome 16 bac clone cit987sk-a-234f9,eif3s8,human translation initiation factor eif-3 p110 subunit gene, complete cds

Total number of rows: 36864

Table truncated, full table size 3452 Kbytes.






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