PMID- 12799456
OWN - NLM
STAT- MEDLINE
DCOM- 20030703
LR  - 20191210
IS  - 1362-4962 (Electronic)
IS  - 0305-1048 (Print)
IS  - 0305-1048 (Linking)
VI  - 31
IP  - 12
DP  - 2003 Jun 15
TI  - Optimization of high-density cDNA-microarray protocols by 'design of 
      experiments'.
PG  - e67
AB  - Expression analysis using microarray technology implies a complex experimental 
      procedure with a large number of parameters affecting the final result. We have 
      demonstrated that optimization of such a complex protocol can be far better 
      handled using design of experiments (DOE) than by working on a single parameter 
      at a time. Based on the results of a screening design, we developed a spotting 
      buffer composed of formamide, betaine and nitrocellulose. This buffer provides a 
      2-fold increase in signal-to-background ratio compared to 3x SSC. Comparison to 
      seven other buffers tested on 10 different substrates revealed it had the highest 
      sensitivity. DNA dissolved in this buffer can be spotted on epoxysilane-coated 
      microscope slides at a density of up to 70 000 spots per slide. A second DOE 
      approach characterized the RNA labeling process with regard to the concentration 
      of fluorescent dyes, dNTPs and reverse transcriptase. Adjust ments of the 
      concentrations of dNTPs, as well as reverse transcriptase, towards the optimum, 
      produced an improvement in the performance of the labeling procedure by a factor 
      of 3 (Cy3) and 10 (Cy5). These results demonstrate that the process of 
      establishing a stable expression profiling protocol and its further optimization 
      can be significantly shortened and improved by DOE.
FAU - Wrobel, Gunnar
AU  - Wrobel G
AD  - Division of Molecular Genetics, Deutsches Krebsforschungszentrum, Im Neuenheimer 
      Feld 280, D-69120 Heidelberg, Germany.
FAU - Schlingemann, Joerg
AU  - Schlingemann J
FAU - Hummerich, Lars
AU  - Hummerich L
FAU - Kramer, Heidi
AU  - Kramer H
FAU - Lichter, Peter
AU  - Lichter P
FAU - Hahn, Meinhard
AU  - Hahn M
LA  - eng
PT  - Evaluation Study
PT  - Journal Article
PT  - Research Support, Non-U.S. Gov't
PL  - England
TA  - Nucleic Acids Res
JT  - Nucleic acids research
JID - 0411011
RN  - 0 (Buffers)
RN  - 0 (Oligonucleotide Probes)
RN  - 63231-63-0 (RNA)
RN  - 9007-49-2 (DNA)
SB  - IM
EIN - Nucleic Acids Res. 2003 Dec;31(23):7057
MH  - Buffers
MH  - DNA/chemistry
MH  - Gene Expression Profiling/*methods
MH  - HL-60 Cells
MH  - Humans
MH  - Oligonucleotide Array Sequence Analysis/*methods
MH  - Oligonucleotide Probes
MH  - RNA/analysis
PMC - PMC162342
EDAT- 2003/06/12 05:00
MHDA- 2003/07/04 05:00
PMCR- 2003/06/15
CRDT- 2003/06/12 05:00
PHST- 2003/06/12 05:00 [pubmed]
PHST- 2003/07/04 05:00 [medline]
PHST- 2003/06/12 05:00 [entrez]
PHST- 2003/06/15 00:00 [pmc-release]
AID - gng067 [pii]
AID - 10.1093/nar/gng067 [doi]
PST - ppublish
SO  - Nucleic Acids Res. 2003 Jun 15;31(12):e67. doi: 10.1093/nar/gng067.