Flow Cytometric Quantification of Competitive Reverse Transcription-PCR Products
| Autor: | Wolfgang Göhde, Thomas Pötter, Niels Wedemeyer, Steffi Wetzlich |
|---|---|
| Rok vydání: | 2002 |
| Předmět: |
Immunoblotting
Clinical Biochemistry Population Biology Flow cytometry chemistry.chemical_compound medicine Humans Digoxigenin Lymphocytes Particle Size Microparticle education education.field_of_study medicine.diagnostic_test Reverse Transcriptase Polymerase Chain Reaction Interleukin-8 Biochemistry (medical) Flow Cytometry Molecular biology Reverse transcriptase Reverse transcription polymerase chain reaction chemistry Biotinylation Primer (molecular biology) Dinitrophenols |
| Zdroj: | Clinical Chemistry. 48:1398-1405 |
| ISSN: | 1530-8561 0009-9147 |
| DOI: | 10.1093/clinchem/48.9.1398 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|