title: Big results from small samples: evaluation of amplification protocols for gene expression profiling.
abstract: Microarrays have revolutionized many areas of biology due to our technical ability to quantify tens of thousands of transcripts within a single experiment. However, there are still many areas that cannot benefit from this technology due to the amount of biological material needed for microarray analysis. In response to this demand, chemistries have been developed that boast the capability of generating targets from nanogram amounts of total RnA, reflecting minimal amounts of biological material, on the order of several hundred or thousand cells. Herein, we describe the evaluation of four chemistries for RnA amplification in terms of reproducibility, sensitivity, accuracy, and comparability to results from a single round of T7 amplification. No evidence for false-positive measurements of differential expression was observed. In contrast, clear differences between chemistries in sensitivity and accuracy were detected. PCR validation showed an interaction of probe sequence on the array and target labeling chemistry, resulting in a chemistry-dependent probe set sensitivity varying over an order of magnitude.
authors.affiliation: Bionomics Research and Technology Center, University of Medicine and Dentistry of New Jersey, Piscataway, New Jersey 08857, USA.
authors.forename: Agnes
authors.initials: A
authors.lastname: Viale
authors.forename: Juan
authors.initials: J
authors.lastname: Li
authors.forename: Jay
authors.initials: J
authors.lastname: Tiesman
authors.forename: Susan
authors.initials: S
authors.lastname: Hester
authors.forename: Aldo
authors.initials: A
authors.lastname: Massimi
authors.forename: Chandi
authors.initials: C
authors.lastname: Griffin
authors.forename: George
authors.initials: G
authors.lastname: Grills
authors.forename: Greg
authors.initials: G
authors.lastname: Khitrov
authors.forename: Kathryn
authors.initials: K
authors.lastname: Lilley
authors.forename: Kevin
authors.initials: K
authors.lastname: Knudtson
authors.forename: Bill
authors.initials: B
authors.lastname: Ward
authors.forename: Karl
authors.initials: K
authors.lastname: Kornacker
authors.forename: Chin-Yi
authors.initials: CY
authors.lastname: Chu
authors.forename: Herbert
authors.initials: H
authors.lastname: Auer
authors.forename: Andrew I
authors.initials: AI
authors.lastname: Brooks
mesh_terms: D045744:Cell Line, Tumor; D020869:Gene Expression Profiling / Q000379:methods*; D006801:Humans; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D018401:Sample Size; D012680:Sensitivity and Specificity