A22
Whole transcriptome amplification from single-cell amounts of total RNA or direct cell lysates and analysis by microarrays
Leah Turner, Pengchin Chen, Paulina Walichiewicz, Victor Sementchenko, Garrett Miyada, Savina Adamo
NuGEN Technologies, San Carlos, USA
Background
The elucidation of gene expression patterns is technically challenging when dealing with very small amounts of RNA, such as can be expected from a few cells. Characterisation of pathways on valuable homogenous samples such as circulating tumour and stem cells, as well as those obtained through laser-capture microscopy, are severely handicapped without a robust amplification system. We describe results obtained from a model LPS induction system that demonstrates whole transcriptome analysis from RNA amounts as low as a single cell. The amplification is based on NuGENs linear isothermal amplification technology that has been optimized to start with single-cell equivalents of purified RNA or from direct cell lysates.
Method
THP-1 human monocytes were induced by LPS. Isolated RNA (10 pg) or cell lysates (5 and 20 cells) were amplified with NuGENs WT-Ovation One-Direct Amplification System. The resulting material was hybridized on GeneChip Gene ST arrays and gene expression patterns were determined.
Results
Total RNA and direct cell lysates resulted in at least 4 μg of amplified material, which was then processed and hybridized on microarrays. Treated and untreated samples were clearly differentiated by Principle Component Analysis. The resulting expression data demonstrated that LPS exposure resulted in induction of genes involved with the inflammatory response. Cytokine and chemokine gene expression levels were increased after a 4-5 hr exposure to LPS when compared to an untreated control.
Conclusion
We have demonstrated that whole transcriptome expression patterns can be obtained from samples as low as a single cell by using the model system described above. The ability to reliably obtain gene expression from homogeneous but limited cell numbers will enable greater insights into cancer-specific pathways and hold the promise of discovery of new therapeutic markers.
