Author:
Nygaard Vigdis,Holden Marit,Løland Anders,Langaas Mette,Myklebost Ola,Hovig Eivind
Abstract
Abstract
Background
Global mRNA amplification has become a widely used approach to obtain gene expression profiles from limited material. An important concern is the reliable reflection of the starting material in the results obtained. This is especially important with extremely low quantities of input RNA where stochastic effects due to template dilution may be present. This aspect remains under-documented in the literature, as quantitative measures of data reliability are most often lacking. To address this issue, we examined the sensitivity levels of each transcript in 3 different cell sample sizes. ANOVA analysis was used to estimate the overall effects of reduced input RNA in our experimental design. In order to estimate the validity of decreasing sample sizes, we examined the sensitivity levels of each transcript by applying a novel model-based method, TransCount.
Results
From expression data, TransCount provided estimates of absolute transcript concentrations in each examined sample. The results from TransCount were used to calculate the Pearson correlation coefficient between transcript concentrations for different sample sizes. The correlations were clearly transcript copy number dependent. A critical level was observed where stochastic fluctuations became significant. The analysis allowed us to pinpoint the gene specific number of transcript templates that defined the limit of reliability with respect to number of cells from that particular source. In the sample amplifying from 1000 cells, transcripts expressed with at least 121 transcripts/cell were statistically reliable and for 250 cells, the limit was 1806 transcripts/cell. Above these thresholds, correlation between our data sets was at acceptable values for reliable interpretation.
Conclusion
These results imply that the reliability of any amplification experiment must be validated empirically to justify that any gene exists in sufficient quantity in the input material. This finding has important implications for any experiment where only extremely small samples such as single cell analyses or laser captured microdissected cells are available.
Publisher
Springer Science and Business Media LLC
Reference54 articles.
1. Assersohn L, Gangi L, Zhao Y, Dowsett M, Simon R, Powles TJ, Liu ET: The Feasibility of Using Fine Needle Aspiration from Primary Breast Cancers for cDNA Microarray Analyses. Clin Cancer Res. 2002, 8: 794-801.
2. Van Gelder RN, von Zastrow ME, Yool A, Dement WC, Barchas JD, Eberwine JH: Amplified RNA synthesized from limited quantities of heterogeneous cDNA. Proc Natl Acad Sci U S A. 1990, 87: 1663-1667.
3. Wang E, Miller LD, Ohnmacht GA, Liu ET, Marincola FM: High-fidelity mRNA amplification for gene profiling. Nat Biotechnol. 2000, 18: 457-459. 10.1038/74546.
4. Baugh LR, Hill AA, Brown EL, Hunter CP: Quantitative analysis of mRNA amplification by in vitro transcription. Nucleic Acids Res. 2001, 29: E29-10.1093/nar/29.5.e29.
5. Iscove NN, Barbara M, Gu M, Gibson M, Modi C, Winegarden N: Representation is faithfully preserved in global cDNA amplified exponentially from sub-picogram quantities of mRNA. Nat Biotechnol. 2002
Cited by
35 articles.
订阅此论文施引文献
订阅此论文施引文献,注册后可以免费订阅5篇论文的施引文献,订阅后可以查看论文全部施引文献