Abstract
HiCEP is a method for gene expression profiling that is based on the AFLPmethod and doesn't require any sequence information for its analysis.Compared to pre-existing similar procedures its false positive rate is verylow, enabling us to assign almost all signals to specific transcripts.HiCEP uses competitive PCR technology and is extremely sensitive, detectingeven one copy / cell and less than 1.5-fold expression changes.Here we have attempted to minimize the starting material required for HiCEPanalysis. The standard protocol requires approximately 1.0 to 2.0micrograms of messenger RNA for one analysis. We have been able to reducethis to 10 nanograms of total RNA. This is equivalent to the amount inabout 1,000 mammalian cells and is [0.0001-0.001%] 1/1,000-10,000 of theamount required using the standard protocol. We observed that thesensitivity clearly depends on the amount of starting material and thatstarting material is particularly scarce for rarely-expressed transcripts.Currently we are getting results using only 100 cells, but the sensitivityat this level needs improvement.
