Application of four dyes in gene expression analyses by microarrays.
BACKGROUND
DNA microarrays are widely used in gene expression analyses. To increase throughput and minimize costs without reducing gene expression data obtained, we investigated whether four mRNA samples can be analyzed simultaneously by applying four different fluorescent dyes.


RESULTS
Following tests for cross-talk of fluorescence signals, Alexa 488, Alexa 594, Cyanine 3 and Cyanine 5 were selected for hybridizations. For self-hybridizations, a single RNA sample was labelled with all dyes and hybridized on commercial cDNA arrays or on in-house spotted oligonucleotide arrays. Correlation coefficients for all combinations of dyes were above 0.9 on the cDNA array. On the oligonucleotide array they were above 0.8, except combinations with Alexa 488, which were approximately 0.5. Standard deviation of expression differences for replicate spots were similar on the cDNA array for all dye combinations, but on the oligonucleotide array combinations with Alexa 488 showed a higher variation.


CONCLUSIONS
In conclusion, the four dyes can be used simultaneously for gene expression experiments on the tested cDNA array, but only three dyes can be used on the tested oligonucleotide array. This was confirmed by hybridizations of control with test samples, as all combinations returned similar numbers of differentially expressed genes with comparable effects on gene expression.
Staal
Yvonne C M
YC
Department of Health Risk Analysis and Toxicology, Maastricht University, 6200 MD Maastricht, The Netherlands. y.staal@grat.unimaas.nl
van Herwijnen
Marcel H M
MH
van Schooten
Frederik J
FJ
van Delft
Joost H M
JH
D001564:Benzo(a)pyrene / Q000494:pharmacology; D002460:Cell Line; D003001:Cloning, Molecular; D004396:Coloring Agents / Q000494:pharmacology; D015342:DNA Probes; D018076:DNA, Complementary / Q000378:metabolism; D005456:Fluorescent Dyes / Q000494:pharmacology*; D015870:Gene Expression; D020869:Gene Expression Profiling / Q000295:instrumentation / Q000379:methods*; D006801:Humans; D006834:Hydrazines / Q000494:pharmacology; D007834:Lasers; D009693:Nucleic Acid Hybridization; D020411:Oligonucleotide Array Sequence Analysis / Q000295:instrumentation / Q000379:methods*; D015345:Oligonucleotide Probes / Q000737:chemistry; D009841:Oligonucleotides / Q000737:chemistry; D012313:RNA / Q000737:chemistry; D012044:Regression Analysis; D012984:Software; D013194:Staining and Labeling / Q000379:methods*; D013997:Time Factors; D014158:Transcription, Genetic
C417561:Alexa 488 hydrazide; D004396:Coloring Agents; D015342:DNA Probes; D018076:DNA, Complementary; D005456:Fluorescent Dyes; D006834:Hydrazines; D015345:Oligonucleotide Probes; D009841:Oligonucleotides; D001564:Benzo(a)pyrene; D012313:RNA