Laser capture microdissection and single-cell RT-PCR without RNA purification.
Chronic infectious diseases of the central nervous system (CNS) are characterized by intrathecal synthesis of increased amounts of immunoglobulin G (IgG) directed against the agent that causes disease. In other inflammatory CNS diseases such as multiple sclerosis and CNS sarcoid, the targets of the humoral immune response are uncertain. To identify the IgGs expressed by individual CD38(+) plasma cells seen in human brain sections, we merged the techniques of laser capture microdissection (LCM) and single-cell RT-PCR. Frozen brain sections from a patient who died of subacute sclerosing panencephalitis (SSPE), were rapidly immunostained and examined by LCM to dissect individual CD38(+) cells. After cell lysis, we developed two techniques for reverse-transcription (RT) of unpurified total RNA in the cell lysates. The first method performed repeated and rapid freeze-thawing, followed by centrifugation of the cell lysate into tubes for subsequent RT. The second, more successful method performed RT in situ on detergent-solubilized cells directly on the cap surface; subsequent nested PCR identified heavy and light chain sequences expressed by two-thirds of individually isolated plasma cells. These techniques will streamline the identification of gene expression products in single cells from complex tissues and have the potential to identify IgGs expressed in the CNS of inflammatory diseases of unknown etiology.
Keays
Kathryne Melissa
KM
Department of Neurology, University of Colorado Health Sciences Center, 4200 East 9th Avenue, Mail Stop B182, Denver, CO 80262, United States.
Owens
Gregory P
GP
Ritchie
Alanna M
AM
Gilden
Donald H
DH
Burgoon
Mark P
MP
D036541:ADP-ribosyl Cyclase / Q000276:immunology; D051997:ADP-ribosyl Cyclase 1; D015703:Antigens, CD / Q000276:immunology; D006801:Humans; D007074:Immunoglobulin G / Q000235:genetics / Q000276:immunology; D007834:Lasers; D008562:Membrane Glycoproteins; D042282:Microdissection / Q000379:methods*; D010950:Plasma Cells / Q000276:immunology; D012333:RNA, Messenger; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*
D015703:Antigens, CD; D007074:Immunoglobulin G; D008562:Membrane Glycoproteins; D012333:RNA, Messenger; D036541:ADP-ribosyl Cyclase; C498154:CD38 protein, human; D051997:ADP-ribosyl Cyclase 1