Flow cytometric quantification of competitive reverse transcription-PCR products.
BACKGROUND
Competitive PCR of reverse transcribed mRNA sequences is used to quantify transcripts, but the usual approaches are labor-intensive and time-consuming. We describe the non-gel-based quantification of competitive reverse transcription (RT)-PCR products with use of microparticles and flow cytometry.


METHODS
PCR products of a target sequence and an internal control sequence (competitor) were labeled during PCR using digoxigenin (DIG)- and dinitrophenol (DNP)-labeled primer, respectively, allowing specific binding to microparticles coated with the corresponding antibody. Both amplification products were biotinylated to enable fluorescence labeling with streptavidin-R-phycoerythrin. The mean fluorescence intensity of each microparticle population, corresponding to the amount of bound PCR product, was measured in a flow cytometer. We constructed microparticles coated with antibodies against DIG and DNP to specifically capture PCR products derived from target and competitor sequences, respectively.


RESULTS
As required for a reliable competitive PCR assay, nearly identical kinetics were found for the amplification of target and competitor sequences when using only one competitive primer. The method was applied to examine interleukin-8 expression in human lymphocytes after x-irradiation. One hour after irradiation, the concentration of transcripts decreased by half.


CONCLUSIONS
The flow cytometric assay for the quantification of competitive RT-PCR products avoids additional hybridization steps and antibody labeling. The use of paramagnetic microparticles would also enable the complete automation of this method.
Wedemeyer
Niels
N
Department of Radiation Biology, Westfälische Wilhelms-Universität Münster, Robert-Koch-Strasse 43, 48149 Münster, Germany. wedemey@uni-muenster.de
Pötter
Thomas
T
Wetzlich
Steffi
S
Göhde
Wolfgang
W
D004076:Digoxigenin / Q000737:chemistry; D004140:Dinitrophenols / Q000737:chemistry; D005434:Flow Cytometry; D006801:Humans; D015151:Immunoblotting; D016209:Interleukin-8 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008214:Lymphocytes / Q000378:metabolism / Q000528:radiation effects; D010316:Particle Size; D020133:Reverse Transcriptase Polymerase Chain Reaction
D004140:Dinitrophenols; D016209:Interleukin-8; D004076:Digoxigenin