Big results from small samples: evaluation of amplification protocols for gene expression profiling.
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.
Viale
Agnes
A
Bionomics Research and Technology Center, University of Medicine and Dentistry of New Jersey, Piscataway, New Jersey 08857, USA.
Li
Juan
J
Tiesman
Jay
J
Hester
Susan
S
Massimi
Aldo
A
Griffin
Chandi
C
Grills
George
G
Khitrov
Greg
G
Lilley
Kathryn
K
Knudtson
Kevin
K
Ward
Bill
B
Kornacker
Karl
K
Chu
Chin-Yi
CY
Auer
Herbert
H
Brooks
Andrew I
AI
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