Miniaturized fluorescent RNA dot blot method for rapid quantitation of gene expression.
BACKGROUND
RNA dot blot hybridization is a commonly used technique for gene expression assays. However, membrane based RNA dot/slot blot hybridization is time consuming, requires large amounts of RNA, and is less suited for parallel assays of more than one gene at a time. Here, we describe a glass-slide based miniaturized RNA dot blot (RNA array) procedure for rapid and parallel gene expression analysis using fluorescently labeled probes.


RESULTS
RNA arrays were prepared by simple manual spotting of RNA onto amino-silane coated microarray glass slides, and used for two-color fluorescent hybridization with specific probes labeled with Cy3 and 18S ribosomal RNA house-keeping gene probe labeled with Cy5 fluorescent dyes. After hybridization, arrays were scanned on a fluorescent microarray scanner and images analyzed using microarray image analysis software. We demonstrate that this method gives comparable results to Northern blot analysis, and enables high throughput quantification of transcripts from nanogram quantities of total RNA in hundreds of samples.


CONCLUSIONS
RNA array on glass slide and detection by fluorescently labeled probes can be used for rapid and parallel gene expression analysis. The method is particularly well suited for gene expression assays that involve quantitation of many transcripts in large numbers of samples.
Yadetie
Fekadu
F
Department of Cancer Research and Molecular Medicine, Faculty of Medicine, Norwegian University of Science and Technology, N-7489 Trondheim, Norway. fekadu.yadetie@medisin.ntnu.no
Sandvik
Arne K
AK
Bergum
Hallgeir
H
Norsett
Kristin
K
Laegreid
Astrid
A
D000818:Animals; D015152:Blotting, Northern / Q000379:methods; D002995:Clofibric Acid / Q000031:analogs & derivatives* / Q000494:pharmacology; D058607:Fibric Acids; D005456:Fluorescent Dyes / Q000737:chemistry*; D005765:Gastrointestinal Agents / Q000494:pharmacology; D015870:Gene Expression / Q000187:drug effects; D000960:Hypolipidemic Agents / Q000494:pharmacology; D007091:Image Processing, Computer-Assisted; D008099:Liver / Q000737:chemistry / Q000187:drug effects; D008832:Microchemistry / Q000379:methods; D008904:Miniaturization / Q000379:methods*; D015336:Molecular Probe Techniques; D009693:Nucleic Acid Hybridization / Q000379:methods; D015282:Octreotide / Q000494:pharmacology; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D012313:RNA / Q000737:chemistry*; D012333:RNA, Messenger / Q000737:chemistry; D051381:Rats; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013997:Time Factors
D058607:Fibric Acids; D005456:Fluorescent Dyes; D005765:Gastrointestinal Agents; D000960:Hypolipidemic Agents; D012333:RNA, Messenger; D002995:Clofibric Acid; D012313:RNA; C019304:ciprofibrate; D015282:Octreotide