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Laser capture microdissection and single-cell RT-PCR without RNA purification
Kathryne Melissa Keays1, Gregory P Owens, Alanna M Ritchie
1Department of Neurology, University of Colorado Health Sciences Center, 4200 East 9th Avenue, Mail Stop B182, Denver, CO 80262, United States.
Insights
Researchers developed a new method to identify specific antibodies (immunoglobulin G) in individual immune cells within the central nervous system (CNS). This technique aids in understanding immune responses in neurological diseases with unknown causes.
Area of Science:
- Neurology
- Immunology
- Molecular Biology
Background:
- Chronic central nervous system (CNS) infections involve increased immunoglobulin G (IgG) synthesis against the causative agent.
- The specific targets of humoral immune responses in inflammatory CNS diseases like multiple sclerosis and sarcoidosis remain unclear.
Purpose of the Study:
- To identify specific immunoglobulin G (IgG) molecules produced by individual CD38(+) plasma cells in human brain tissue.
- To develop and refine techniques for analyzing gene expression in single cells from complex tissues.
Main Methods:
- Combined laser capture microdissection (LCM) with single-cell reverse-transcription polymerase chain reaction (RT-PCR).
- Developed two methods for reverse-transcription (RT) of unpurified total RNA from single-cell lysates.
- Optimized an in situ RT method on detergent-solubilized cells followed by nested PCR to identify heavy and light chain sequences.
Main Results:
- Successfully identified heavy and light chain sequences of immunoglobulin G (IgG) in two-thirds of individually isolated plasma cells.
- The in situ RT method proved more successful than freeze-thaw techniques for RNA analysis.
- Demonstrated the feasibility of analyzing gene expression products in single cells from complex tissues.
Conclusions:
- The developed techniques streamline the identification of gene expression products in single cells.
- This approach has significant potential for identifying specific immunoglobulin G (IgG) targets in CNS inflammatory diseases of unknown etiology.
- Advances understanding of humoral immunity in neurological disorders.
Abstract:
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.
