title: Effects of sample size on differential gene expression, rank order and prediction accuracy of a gene signature.
abstract: Top differentially expressed gene lists are often inconsistent between studies and it has been suggested that small sample sizes contribute to lack of reproducibility and poor prediction accuracy in discriminative models. We considered sex differences (69♂, 65 ♀) in 134 human skeletal muscle biopsies using DNA microarray. The full dataset and subsamples (n = 10 (5 ♂, 5 ♀) to n = 120 (60 ♂, 60 ♀)) thereof were used to assess the effect of sample size on the differential expression of single genes, gene rank order and prediction accuracy. Using our full dataset (n = 134), we identified 717 differentially expressed transcripts (p<0.0001) and we were able predict sex with ~90% accuracy, both within our dataset and on external datasets. Both p-values and rank order of top differentially expressed genes became more variable using smaller subsamples. For example, at n = 10 (5 ♂, 5 ♀), no gene was considered differentially expressed at p<0.0001 and prediction accuracy was ~50% (no better than chance). We found that sample size clearly affects microarray analysis results; small sample sizes result in unstable gene lists and poor prediction accuracy. We anticipate this will apply to other phenotypes, in addition to sex.
authors.affiliation: Department of Oncology, University of Alberta, Cross Cancer Institute, Edmonton, Alberta, Canada.
authors.forename: Cynthia
authors.initials: C
authors.lastname: Stretch
authors.forename: Sheehan
authors.initials: S
authors.lastname: Khan
authors.forename: Nasimeh
authors.initials: N
authors.lastname: Asgarian
authors.forename: Roman
authors.initials: R
authors.lastname: Eisner
authors.forename: Saman
authors.initials: S
authors.lastname: Vaisipour
authors.forename: Sambasivarao
authors.initials: S
authors.lastname: Damaraju
authors.forename: Kathryn
authors.initials: K
authors.lastname: Graham
authors.forename: Oliver F
authors.initials: OF
authors.lastname: Bathe
authors.forename: Helen
authors.initials: H
authors.lastname: Steed
authors.forename: Russell
authors.initials: R
authors.lastname: Greiner
authors.forename: Vickie E
authors.initials: VE
authors.lastname: Baracos
mesh_terms: D000368:Aged; D005260:Female; D014644:Genetic Variation; D006801:Humans; D008297:Male; D008875:Middle Aged; D009369:Neoplasms / Q000235:genetics / Q000473:pathology / Q000601:surgery; D020411:Oligonucleotide Array Sequence Analysis / Q000592:standards / Q000706:statistics & numerical data*; D011237:Predictive Value of Tests; D012333:RNA, Messenger / Q000032:analysis* / Q000235:genetics; D017568:Rectus Abdominis / Q000737:chemistry* / Q000378:metabolism; D015203:Reproducibility of Results; D018401:Sample Size; D059467:Transcriptome
chemical_list: D012333:RNA, Messenger