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Do-it-yourself: construction of a custom cDNA macroarray platform with high sensitivity and linear range

Tom Boonefaes, Erica Houthuys UGent, Rafael Van den Bergh, Seppe Vander Beken UGent, Geert Raes, Peter Brouckaert UGent, P De Baetselier and Johan Grooten UGent (2011) BMC BIOTECHNOLOGY. 11.
abstract
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 mu g 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.
Please use this url to cite or link to this publication:
author
organization
year
type
journalArticle (original)
publication status
published
subject
keyword
CONTROL MAQC PROJECT, GENE-EXPRESSION, RNA AMPLIFICATION, MICROARRAY, PERFORMANCE, MOUSE
journal title
BMC BIOTECHNOLOGY
BMC Biotechnol.
volume
11
article_number
97
pages
12 pages
Web of Science type
Article
Web of Science id
000297045200001
JCR category
BIOTECHNOLOGY & APPLIED MICROBIOLOGY
JCR impact factor
2.349 (2011)
JCR rank
69/157 (2011)
JCR quartile
2 (2011)
ISSN
1472-6750
DOI
10.1186/1472-6750-11-97
language
English
UGent publication?
yes
classification
A1
copyright statement
I have retained and own the full copyright for this publication
id
1963732
handle
http://hdl.handle.net/1854/LU-1963732
date created
2011-12-08 14:56:48
date last changed
2012-06-26 14:32:30
@article{1963732,
  abstract     = {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 {\textlangle} 0.0001). Furthermore, the assay has low cost and input RNA requirements (0.5 mu g 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.},
  articleno    = {97},
  author       = {Boonefaes, Tom and Houthuys, Erica and Van den Bergh, Rafael and Vander Beken, Seppe and Raes, Geert and Brouckaert, Peter and De Baetselier, P and Grooten, Johan},
  issn         = {1472-6750},
  journal      = {BMC BIOTECHNOLOGY},
  keyword      = {CONTROL MAQC PROJECT,GENE-EXPRESSION,RNA AMPLIFICATION,MICROARRAY,PERFORMANCE,MOUSE},
  language     = {eng},
  pages        = {12},
  title        = {Do-it-yourself: construction of a custom cDNA macroarray platform with high sensitivity and linear range},
  url          = {http://dx.doi.org/10.1186/1472-6750-11-97},
  volume       = {11},
  year         = {2011},
}

Chicago
Boonefaes, Tom, Erica Houthuys, Rafael Van den Bergh, Seppe Vander Beken, Geert Raes, Peter Brouckaert, P De Baetselier, and Johan Grooten. 2011. “Do-it-yourself: Construction of a Custom cDNA Macroarray Platform with High Sensitivity and Linear Range.” Bmc Biotechnology 11.
APA
Boonefaes, T., Houthuys, E., Van den Bergh, R., Vander Beken, S., Raes, G., Brouckaert, P., De Baetselier, P., et al. (2011). Do-it-yourself: construction of a custom cDNA macroarray platform with high sensitivity and linear range. BMC BIOTECHNOLOGY, 11.
Vancouver
1.
Boonefaes T, Houthuys E, Van den Bergh R, Vander Beken S, Raes G, Brouckaert P, et al. Do-it-yourself: construction of a custom cDNA macroarray platform with high sensitivity and linear range. BMC BIOTECHNOLOGY. 2011;11.
MLA
Boonefaes, Tom, Erica Houthuys, Rafael Van den Bergh, et al. “Do-it-yourself: Construction of a Custom cDNA Macroarray Platform with High Sensitivity and Linear Range.” BMC BIOTECHNOLOGY 11 (2011): n. pag. Print.