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RNA pre-amplification enables large-scale RT-qPCR gene-expression studies on limiting sample amounts

Joëlle Vermeulen UGent, Stefaan Derveaux UGent, Steve Lefever UGent, Els De Smet UGent, Katleen De Preter UGent, Nurten Yigit UGent, Anne De Paepe UGent, Filip Pattyn UGent, Franki Speleman UGent and Jo Vandesompele UGent (2009) BMC RESEARCH NOTES. 2.
abstract
Background : The quantitative polymerase chain reaction (qPCR) is a widely utilized method for gene-expression analysis. However, insufficient material often compromises large-scale gene-expression studies. The aim of this study is to evaluate an RNA pre-amplification method to produce micrograms of cDNA as input for qPCR. Findings : The linear isothermal Ribo-SPIA pre-amplification method (WT-Ovation; NuGEN) was first evaluated by measuring the expression of 20 genes in RNA samples from six neuroblastoma cell lines and of 194 genes in two commercially available reference RNA samples before and after pre-amplification, and subsequently applied on a large panel of 738 RNA samples extracted from neuroblastoma tumours. All RNA samples were evaluated for RNA integrity and purity. Starting from 5 to 50 nanograms of total RNA the sample pre-amplification method was applied, generating approximately 5 microgams of cDNA, sufficient to measure more than 1000 target genes. The results obtained from this study show a constant yield of pre-amplified cDNA independent of the amount of input RNA; preservation of differential gene-expression after pre-amplification without introduction of substantial bias; no co-amplification of contaminating genomic DNA; no necessity to purify the pre-amplified material; and finally the importance of good RNA quality to enable pre-amplification. Conclusion : Application of this unbiased and easy to use sample pre-amplification technology offers great advantage to generate sufficient material for diagnostic and prognostic work-up and enables large-scale qPCR gene-expression studies using limited amounts of sample material.
Please use this url to cite or link to this publication:
author
organization
year
type
journalArticle (original)
publication status
published
subject
journal title
BMC RESEARCH NOTES
BMC Res. Notes
volume
2
article_number
235
pages
9 pages
ISSN
1756-0500
DOI
10.1186/1756-0500-2-235
language
English
UGent publication?
yes
classification
A2
copyright statement
I have retained and own the full copyright for this publication
VABB id
c:vabb:282058
VABB type
VABB-1
id
837424
handle
http://hdl.handle.net/1854/LU-837424
date created
2010-01-25 15:48:08
date last changed
2015-06-17 11:20:41
@article{837424,
  abstract     = {Background : The quantitative polymerase chain reaction (qPCR) is a widely utilized method for gene-expression analysis. However, insufficient material often compromises large-scale gene-expression studies. The aim of this study is to evaluate an RNA pre-amplification method to produce micrograms of cDNA as input for qPCR.
Findings : The linear isothermal Ribo-SPIA pre-amplification method (WT-Ovation; NuGEN) was first evaluated by measuring the expression of 20 genes in RNA samples from six neuroblastoma cell lines and of 194 genes in two commercially available reference RNA samples before and after pre-amplification, and subsequently applied on a large panel of 738 RNA samples extracted from neuroblastoma tumours. All RNA samples were evaluated for RNA integrity and purity. Starting from 5 to 50 nanograms of total RNA the sample pre-amplification method was applied, generating approximately 5 microgams of cDNA, sufficient to measure more than 1000 target genes. The results obtained from this study show a constant yield of pre-amplified cDNA independent of the amount of input RNA; preservation of differential gene-expression after pre-amplification without introduction of substantial bias; no co-amplification of contaminating genomic DNA; no necessity to purify the pre-amplified material; and finally the importance of good RNA quality to enable pre-amplification. Conclusion : Application of this unbiased and easy to use sample pre-amplification technology offers great advantage to generate sufficient material for diagnostic and prognostic work-up and enables large-scale qPCR gene-expression studies using limited amounts of sample material.},
  articleno    = {235},
  author       = {Vermeulen, Jo{\"e}lle and Derveaux, Stefaan and Lefever, Steve and De Smet, Els and De Preter, Katleen and Yigit, Nurten and De Paepe, Anne and Pattyn, Filip and Speleman, Franki and Vandesompele, Jo},
  issn         = {1756-0500},
  journal      = {BMC RESEARCH NOTES},
  language     = {eng},
  pages        = {9},
  title        = {RNA pre-amplification enables large-scale RT-qPCR gene-expression studies on limiting sample amounts},
  url          = {http://dx.doi.org/10.1186/1756-0500-2-235},
  volume       = {2},
  year         = {2009},
}

Chicago
Vermeulen, Joëlle, Stefaan Derveaux, Steve Lefever, Els De Smet, Katleen De Preter, Nurten Yigit, Anne De Paepe, Filip Pattyn, Franki Speleman, and Jo Vandesompele. 2009. “RNA Pre-amplification Enables Large-scale RT-qPCR Gene-expression Studies on Limiting Sample Amounts.” Bmc Research Notes 2.
APA
Vermeulen, Joëlle, Derveaux, S., Lefever, S., De Smet, E., De Preter, K., Yigit, N., De Paepe, A., et al. (2009). RNA pre-amplification enables large-scale RT-qPCR gene-expression studies on limiting sample amounts. BMC RESEARCH NOTES, 2.
Vancouver
1.
Vermeulen J, Derveaux S, Lefever S, De Smet E, De Preter K, Yigit N, et al. RNA pre-amplification enables large-scale RT-qPCR gene-expression studies on limiting sample amounts. BMC RESEARCH NOTES. 2009;2.
MLA
Vermeulen, Joëlle, Stefaan Derveaux, Steve Lefever, et al. “RNA Pre-amplification Enables Large-scale RT-qPCR Gene-expression Studies on Limiting Sample Amounts.” BMC RESEARCH NOTES 2 (2009): n. pag. Print.