chapter  4
7 Pages

Microdissection of Single or Small Numbers of Cells for Analyses of DNA and RNA by PCR

PCR has been used to detect nuclear DNA or reverse transcribed expressed RNA sequences from extracts of various tissues. For many purposes, greater specificity and sensitivity can be achieved if microdissection is used to collect groups of similar normal or pathological cells from tissue sections for nucleic acid extraction. An example of where single cell PCR is required is for the study of somatic

mutations such as in the antibody chains of B lymphocytes,1-7 because the sequences of rearranged DNA for antibody heavy chains of each B cell are unique.1-5 Direct sequencing of amplified DNA from a single cell minimizes errors from in vitro recombination during PCR.8 This would be a particularly serious problem for studies of antibody heavy and light chain sequences in rabbit B cells where blocks of changes occur that are introduced by a gene-conversion-like mechanism.1-4 We started using hydraulic micromanipulation (HM) for single cell collection as described by other laboratories (reviewed in Reference 5). Most of the results we published used cells collected by HM. Since such manual microdissection is extremely difficult and tedious to perform, we turned to laser capture microdissection (LCM). The methods developed to stain and analyze cells for direct comparisons of different microdissection methods are given in detail below. Results of direct comparisons of yields of PCR products and sequences indicative of recovery of a single cell collected by HM or LCM from the same stained sections were published.6 We concluded that the reproducibility, better yields of single cell sequences, greater efficiency, and shorter times needed for both operator training and actual cell collection made LCM a valuable method for single cell collection. In addition to tests of the infrared laser-based LCM instrument manufactured by Arcturus Engineering, we also tested the Leica UV laser-based LMD instrument, doing a similar type of comparison. The ease of use of the noncontact Leica LMD system is very attractive. However, the yields of PCR products and single-cell sequences were unacceptably low.7 On the other hand, isolation of RNA from groups of cells such as the contents of a germinal center in serial sections from spleen was simple and successful by the Leica LMD method (Yang G. et al, unpublished results).