Do-it-yourself: construction of a custom cDNA macroarray platform with high sensitivity and linear range.
BACKGROUND
Research involving gene expression profiling and clinical applications, such as diagnostics and prognostics, often require a DNA array platform that is flexibly customisable and cost-effective, but at the same time is highly sensitive and capable of accurately and reproducibly quantifying the transcriptional expression of a vast number of genes over the whole transcriptome dynamic range using low amounts of RNA sample. Hereto, a set of easy-to-implement practical optimisations to the design of cDNA-based nylon macroarrays as well as sample (33)P-labeling, hybridisation protocols and phosphor screen image processing were analysed for macroarray performance.


RESULTS
The here proposed custom macroarray platform had an absolute sensitivity as low as 50,000 transcripts and a linear range of over 5 log-orders. Its quality of identifying differentially expressed genes was at least comparable to commercially available microchips. Interestingly, the quantitative accuracy was found to correlate significantly with corresponding reversed transcriptase - quantitative PCR values, the gold standard gene expression measure (Pearson's correlation test p < 0.0001). Furthermore, the assay has low cost and input RNA requirements (0.5 μg and less) and has a sound reproducibility.


CONCLUSIONS
Results presented here, demonstrate for the first time that self-made cDNA-based nylon macroarrays can produce highly reliable gene expression data with high sensitivity and covering the entire mammalian dynamic range of mRNA abundances. Starting off from minimal amounts of unamplified total RNA per sample, a reasonable amount of samples can be assayed simultaneously for the quantitative expression of hundreds of genes in an easily customisable and cost-effective manner.
Department of Biomedical Molecular Biology, Ghent University, Belgium.
Tom
T
Boonefaes
Erica
E
Houthuys
Rafael
R
Van den Bergh
Seppe
S
Vander Beken
Geert
G
Raes
Peter
P
Brouckaert
Patrick
P
De Baetselier
Johan
J
Grooten
D017931:DNA Primers; D020869:Gene Expression Profiling / Q000295:instrumentation / Q000379:methods*; D006801:Humans; D008264:Macrophages / Q000378:metabolism*; D009757:Nylons / Q000737:chemistry; D020411:Oligonucleotide Array Sequence Analysis / Q000295:instrumentation / Q000379:methods*; D012333:RNA, Messenger / Q000032:analysis* / Q000096:biosynthesis; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D059467:Transcriptome
D017931:DNA Primers; D009757:Nylons; D012333:RNA, Messenger