title: Limitations of mRNA amplification from small-size cell samples.
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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Department of Tumor Biology, Institute for Cancer Research, The Norwegian Radium Hospital, Montebello, 0310 Oslo, Norway. vigdisny@radium.uio.no
authors.forename: Vigdis
authors.initials: V
authors.lastname: Nygaard
authors.forename: Marit
authors.initials: M
authors.lastname: Holden
authors.forename: Anders
authors.initials: A
authors.lastname: Løland
authors.forename: Mette
authors.initials: M
authors.lastname: Langaas
authors.forename: Ola
authors.initials: O
authors.lastname: Myklebost
authors.forename: Eivind
authors.initials: E
authors.lastname: Hovig
mesh_terms: D000704:Analysis of Variance; D017124:Conserved Sequence; D017931:DNA Primers / Q000737:chemistry; D018076:DNA, Complementary / Q000378:metabolism; D020869:Gene Expression Profiling / Q000379:methods; D005786:Gene Expression Regulation; D015972:Gene Expression Regulation, Neoplastic; D015723:Gene Library; D005821:Genetic Techniques; D006367:HeLa Cells; D006801:Humans; D015233:Models, Statistical; D009693:Nucleic Acid Hybridization; D020411:Oligonucleotide Array Sequence Analysis; D012313:RNA / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism*; D012334:RNA, Neoplasm / Q000378:metabolism; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction
chemical_list: D017931:DNA Primers; D018076:DNA, Complementary; D012333:RNA, Messenger; D012334:RNA, Neoplasm; D012313:RNA