Anne-Marie
Cleton-Jansen, Tom van Wezel, Feija van Bokhoven, Thirza Broekhuizen-van de
Berg, Hans Baelde
Dept.
Pathology, Leiden University Medical Center, Leiden
Labelling
of cDNA for hybridisation is a critical step in the cDNA microarray technique.
The fluorescent labels are expensive and the target cDNA is often unique and
limited. The original method that is applied in Stanford uses micrograms of mRNA
for labelling with Cy5 and Cy3 in a direct reverse transcription, which is an
unfeasible approach for most primary patient samples, simply because it is not
possible to obtain that amount of RNA from such material. In our hands this
method was extremely inefficient because much RNA is lost when purifying mRNA.
We
have tested several protocols for direct incorporation of Cy5 and Cy3 into total
RNA. Using Real Time PCR we assessed which amount of RNA and which reverse
transcription conditions results in the best yield of cDNA.
For
our specific applications direct incorporation was not satisfactory and
therefore we tested several amplification methods. Two types of amplification
can be applied: signal amplification or cDNA amplification. For signal
amplification we have tested two commercially available methods: the Tyramid
Signal Amplification of Perkin Elmer/NEN and the 3DNA Submicro detection kit
from Genisphere. For cDNA amplification we applied in vitro transcription on
double strand cDNA and subsequent direct incorporation of Cy-dyes in a second RT
on the resulting cRNA. Furthermore we have tested the protocol by TIGR for
aminoallyl directed labelling and the ARES Alexa Fluor labelling kit from
Molecular Probes.
In conclusion, few labelling methods give satisfactory results and although companies are supplying kits, these do not always guarantee high signals and low background. Much depends on the quality of the input RNA but also on the coating of the glass slides.