Semiquantitative, nonradioactive RT-PCR detection of immunoglobulin mRNA in human B cells and plasma cells.
Quantification of mRNA is important for studies of gene expression and gene regulation. We investigated the utility of the reverse transcriptase polymerase chain reaction (RT-PCR) approach in the quantification of mRNA from small cell numbers. To take into account the complex kinetics of the PCR amplification process and the nonlinear signal development during detection of PCR products, calibration curves were established on the basis of different, known, starting concentrations of cDNA fragments, different PCR cycle numbers, and different signal intensities. Detection of digoxigenin-labeled PCR products via an enzymatically generated chemiluminescent signal was found to give a reproducible and wider range of signal intensities compared to simple ethidium bromide staining. We applied this methodology to the quantification of immunoglobulin M (IgM) mRNA levels in human B cells. Using an in vitro culture system in which B cells differentiate into plasma cells, the kinetics of IgM mRNA expression were established during a 10-day culture period and a 180-fold mRNA increase was found.
Department of Medicine, University Hospital of Geneva, Switzerland.
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T
Matthes
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V
Kindler
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RH
Zubler
D001402:B-Lymphocytes / Q000166:cytology / Q000378:metabolism*; D001483:Base Sequence; D002138:Calibration; D002454:Cell Differentiation; D002478:Cells, Cultured; D017931:DNA Primers; D018076:DNA, Complementary / Q000235:genetics; D006801:Humans; D007075:Immunoglobulin M / Q000032:analysis* / Q000235:genetics; D007700:Kinetics; D008969:Molecular Sequence Data; D010950:Plasma Cells / Q000166:cytology / Q000378:metabolism*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012333:RNA, Messenger / Q000032:analysis*
D017931:DNA Primers; D018076:DNA, Complementary; D007075:Immunoglobulin M; D012333:RNA, Messenger