Evaluation of the performance of two carbodiimide-based cyanine dyes for detecting changes in mRNA expression with DNA microarrays.
Microarrays have been extensively used to investigate genome-wide expression patterns. Although this technology has been tremendously successful, several practical issues would benefit from improvements in design. Here we describe a novel, efficient labeling methodology that uses carbodiimide-linked cyanine dyes to directly chemically label cDNA derived from mouse total RNA. Using this protocol, it takes only 10 min at 70 degrees C to complete the cDNA labeling reaction. The directly labeled cDNAs can then be hybridized to 70-mer mouse oligonucleotide arrays for expression profiling studies. Microarray analyses indicate that these cDNAs are uniformly labeled and produce higher signal intensities than conventional enzymatic direct labeling methods and comparable signal intensities to those obtained by conventional indirect labeling methods. Furthermore, verification of our microarray data using a reverse transcription PCR (RT-PCR) method indicates good agreement between the two methods. Thus, we conclude that our simplified cyanine-carbodiimide labeling method, which does not rely on the incorporation of modified nucleotides, will provide a reliable, quicker, and potentially cheaper alternative to established labeling techniques for gene expression analyses.
Kimura
Naoki
N
Research and Development Center, Nisshinbo Industries Inc., Chiba, Japan. n-kimu@nisshinbo.co.jp
Tamura
Taka-aki
TA
Murakami
Masahiro
M
D000818:Animals; D002232:Carbocyanines; D002234:Carbodiimides; D018076:DNA, Complementary / Q000737:chemistry* / Q000235:genetics*; D005456:Fluorescent Dyes; D017404:In Situ Hybridization, Fluorescence / Q000379:methods*; D051379:Mice; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D012333:RNA, Messenger / Q000235:genetics* / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D013194:Staining and Labeling / Q000379:methods
D002232:Carbocyanines; D002234:Carbodiimides; D018076:DNA, Complementary; D005456:Fluorescent Dyes; D012333:RNA, Messenger