PMID- 16084216
OWN - NLM
STAT- MEDLINE
DCOM- 20051003
LR  - 20181113
IS  - 0022-1759 (Print)
IS  - 1872-7905 (Electronic)
IS  - 0022-1759 (Linking)
VI  - 302
IP  - 1-2
DP  - 2005 Jul
TI  - Laser capture microdissection and single-cell RT-PCR without RNA purification.
PG  - 90-8
AB  - 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.
FAU - Keays, Kathryne Melissa
AU  - Keays KM
AD  - Department of Neurology, University of Colorado Health Sciences Center, 4200 East 
      9th Avenue, Mail Stop B182, Denver, CO 80262, United States.
FAU - Owens, Gregory P
AU  - Owens GP
FAU - Ritchie, Alanna M
AU  - Ritchie AM
FAU - Gilden, Donald H
AU  - Gilden DH
FAU - Burgoon, Mark P
AU  - Burgoon MP
LA  - eng
GR  - NS41549/NS/NINDS NIH HHS/United States
GR  - NS32623/NS/NINDS NIH HHS/United States
GR  - P01 NS032623/NS/NINDS NIH HHS/United States
GR  - R01 NS041549-04/NS/NINDS NIH HHS/United States
GR  - R01 NS041549/NS/NINDS NIH HHS/United States
PT  - Journal Article
PT  - Research Support, N.I.H., Extramural
PT  - Research Support, U.S. Gov't, P.H.S.
PL  - Netherlands
TA  - J Immunol Methods
JT  - Journal of immunological methods
JID - 1305440
RN  - 0 (Antigens, CD)
RN  - 0 (Immunoglobulin G)
RN  - 0 (Membrane Glycoproteins)
RN  - 0 (RNA, Messenger)
RN  - EC 3.2.2.5 (ADP-ribosyl Cyclase)
RN  - EC 3.2.2.5 (CD38 protein, human)
RN  - EC 3.2.2.6 (ADP-ribosyl Cyclase 1)
SB  - IM
MH  - ADP-ribosyl Cyclase/immunology
MH  - ADP-ribosyl Cyclase 1
MH  - Antigens, CD/immunology
MH  - Humans
MH  - Immunoglobulin G/genetics/immunology
MH  - *Lasers
MH  - Membrane Glycoproteins
MH  - Microdissection/*methods
MH  - Plasma Cells/immunology
MH  - RNA, Messenger
MH  - Reverse Transcriptase Polymerase Chain Reaction/*methods
PMC - PMC3279919
MID - NIHMS353912
EDAT- 2005/08/09 09:00
MHDA- 2005/10/04 09:00
PMCR- 2012/02/15
CRDT- 2005/08/09 09:00
PHST- 2005/02/14 00:00 [received]
PHST- 2005/04/22 00:00 [revised]
PHST- 2005/04/28 00:00 [accepted]
PHST- 2005/08/09 09:00 [pubmed]
PHST- 2005/10/04 09:00 [medline]
PHST- 2005/08/09 09:00 [entrez]
PHST- 2012/02/15 00:00 [pmc-release]
AID - S0022-1759(05)00128-6 [pii]
AID - 10.1016/j.jim.2005.04.018 [doi]
PST - ppublish
SO  - J Immunol Methods. 2005 Jul;302(1-2):90-8. doi: 10.1016/j.jim.2005.04.018.