Limitations of mRNA amplification from small-size cell samples

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

Subject

Genetics,Biotechnology

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

同舟云学术

1.学者识别学者识别

2.学术分析学术分析

3.人才评估人才评估

"同舟云学术"是以全球学者为主线,采集、加工和组织学术论文而形成的新型学术文献查询和分析系统,可以对全球学者进行文献检索和人才价值评估。用户可以通过关注某些学科领域的顶尖人物而持续追踪该领域的学科进展和研究前沿。经过近期的数据扩容,当前同舟云学术共收录了国内外主流学术期刊6万余种,收集的期刊论文及会议论文总量共计约1.5亿篇,并以每天添加12000余篇中外论文的速度递增。我们也可以为用户提供个性化、定制化的学者数据。欢迎来电咨询!咨询电话:010-8811{复制后删除}0370

www.globalauthorid.com

TOP

Copyright © 2019-2024 北京同舟云网络信息技术有限公司
京公网安备11010802033243号  京ICP备18003416号-3