Comparative evaluation of linear and exponential amplification techniques for expression profiling at the single-cell level.
BACKGROUND
Single-cell microarray expression profiling requires 108-109-fold amplification of the picogram amounts of total RNA typically found in eukaryotic cells. Several methods for RNA amplification are in general use, but little consideration has been given to the comparative analysis of those methods in terms of the overall validity of the data generated when amplifying from single-cell amounts of RNA, rather than their empirical performance in single studies.


RESULTS
We tested the performance of three methods for amplifying single-cell amounts of RNA under ideal conditions: T7-based in vitro transcription; switching mechanism at 5' end of RNA template (SMART) PCR amplification; and global PCR amplification. All methods introduced amplification-dependent noise when mRNA was amplified 108-fold, compared with data from unamplified cDNA. PCR-amplified cDNA demonstrated the smallest number of differences between two parallel replicate samples and the best correlation between independent amplifications from the same cell type, with SMART outperforming global PCR amplification. SMART had the highest true-positive rate and the lowest false-positive rate when comparing expression between two different cell types, but had the lowest absolute discovery rate of all three methods. Direct comparison of the performance of SMART and global PCR amplification on single-cell amounts of total RNA and on single neural stem cells confirmed these findings.


CONCLUSIONS
Under the conditions tested, PCR amplification was more reliable than linear amplification for detecting true expression differences between samples. SMART amplification had a higher true-positive rate than global amplification, but at the expense of a considerably lower absolute discovery rate and a systematic compression of observed expression ratios.
Gurdon Institute and Department of Biochemistry, University of Cambridge, Tennis Court Road, Cambridge, CB2 1QN, UK.
Tatiana
T
Subkhankulova
Frederick J
FJ
Livesey
D016475:3T3 Cells; D000818:Animals; D002460:Cell Line; D017931:DNA Primers; D012321:DNA-Directed RNA Polymerases / Q000235:genetics; D005260:Female; D005784:Gene Amplification; D020869:Gene Expression Profiling / Q000379:methods*; D051379:Mice; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods / Q000592:standards; D010053:Ovary; D016133:Polymerase Chain Reaction; D012313:RNA / Q000235:genetics; D015203:Reproducibility of Results; D014158:Transcription, Genetic; D014764:Viral Proteins / Q000235:genetics
D017931:DNA Primers; D014764:Viral Proteins; D012313:RNA; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases