ArticleBMC genomics2003
Amplified RNA degradation in T7-amplification methods results in biased microarray hybridizations.
Article in BMC genomics, 2003. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 19 papers.
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19 citing papers in PubMed, 66 citations in OpenAlex.
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- Development and characterization of a novel method for the analysis of gene expression patterns in lymphatic endothelial cells derived from primary breast tissues.Journal of cancer research and clinical oncology · 2010Article
- Real-time monitoring of aRNA production during T7 amplification to prevent the loss of sample representation during microarray hybridization sample preparation.Nucleic acids research · 2009Article
- "Hook"-calibration of GeneChip-microarrays: chip characteristics and expression measures.Algorithms for molecular biology : AMB · 2008Article
- The expression and bioinformatic analysis of a novel gene C20orf14 associated with lymphoma.Journal of Huazhong University of Science and Technology. Medical sciences = Hua zhong ke ji da xue xue bao. Yi xue Ying De wen ban = Huazhong keji daxue xuebao. Yixue Yingdewen ban · 2008Article
- Amplification biases: possible differences among deviating gene expressions.BMC genomics · 2008Article
- In vitro transcription amplification and labeling methods contribute to the variability of gene expression profiling with DNA microarrays.The Journal of molecular diagnostics : JMD · 2006Article
- Comparative evaluation of linear and exponential amplification techniques for expression profiling at the single-cell level.Genome biology · 2006Article
- Limitations of mRNA amplification from small-size cell samples.BMC genomics · 2005Article
- RNA amplification for successful gene profiling analysis.Journal of translational medicine · 2005Article
- Comparison of standard exponential and linear techniques to amplify small cDNA samples for microarrays.BMC genomics · 2005Article
- Microarray-based comparison of three amplification methods for nanogram amounts of total RNA.American journal of physiology. Cell physiology · 2005Article
- Article
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Authors and funding
3 authors at 1 institution in 1 country.
Funding
No grant is acknowledged in the PubMed record.
Abstract
backgroundThe amplification of RNA with the T7-System is a widely used technique for obtaining increased amounts of RNA starting from limited material. The amplified RNA (aRNA) can subsequently be used for microarray hybridizations, warranting sufficient signal for image analysis. We describe here an amplification-time dependent degradation of aRNA in prolonged standard T7 amplification protocols, that results in lower average size aRNA and decreased yields.
resultsA time-dependent degradation of amplified RNA (aRNA) could be observed when using the classical "Eberwine" T7-Amplification method. When the amplification was conducted for more than 4 hours, the resulting aRNA showed a significantly smaller size distribution on gel electrophoresis and a concomitant reduction of aRNA yield. The degradation of aRNA could be correlated to the presence of the T7 RNA Polymerase in the amplification cocktail. The aRNA degradation resulted in a strong bias in microarray hybridizations with a high coefficient of variation and a significant reduction of signals of certain transcripts, that seem to be susceptible to this RNA degrading activity. The time-dependent degradation of these transcripts was verified by a real-time PCR approach.
conclusionsIt is important to perform amplifications not longer than 4 hours as there is a characteristic 'quality vs. yield' situation for longer amplification times. When conducting microarray hybridizations it is important not to compare results obtained with aRNA from different amplification times.
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