NATURE MEDICINE • VOLUME 5 • NUMBER 1 • JANUARY 1999 117 NEW TECHNOLOGY Gene expression profiles of thousands of genes can now be ex- amined en masse through cDNA and oligonucleotide microar- rays 1–3 . Recently, studies have been reported that examined gene expression changes in yeast 4,5 , as well as in mammalian cell lines 6 , primary cells 7 and tissues 8 . However, present applications of microarray technology do not include the study of gene ex- pression from individual cell types residing in a given tissue/organ (that is, in situ). Such studies would greatly facili- tate our understanding of the complex interactions that exist in vivo between neighboring cell types in normal and disease states. We demonstrate here that gene expression profiles from adjacent cell types can be successfully obtained by integrating the technologies of laser capture microdissection 9 (LCM) and T7-based RNA amplification 10 with cDNA microarrays 11 . Neighboring small and large neurons are individually captured To demonstrate this integration of technologies, we examined the differential gene expression between large- and small-sized neurons in the dorsal root ganglia (DRG). In general, large DRG neurons are myelinated, fast-conducting and transmit mechanosensory information, whereas small neurons are un- myelinated, slow-conducting and transmit nociceptive infor- mation 12 . We chose this system because numerous differentially expressed genes (small versus large) have been reported, thus the success of this experiment could be assessed; and because many small and large neurons are adjacent to each other, thus we could test whether individual neurons can be cleanly cap- tured. Large (diameter of >40 μm) and small (diameter <25 μm and with identified nuclei) neurons were cleanly and individu- ally captured by LCM from sections (10 μm in thickness) of Nissl-stained rat DRG (Fig. 1). For this study, two sets of 1,000 large neurons and three sets of 1,000 small neurons were captured for cDNA microarray analysis. RNA amplification is reproducible between individual captures RNA was extracted from each set of neurons and linearly ampli- fied (independently) an estimated 10 6 -fold using T7 RNA poly- merase. After being amplified, one fluorescently labeled probe was synthesized from an individually amplified RNA (aRNA), di- vided equally into three parts and hybridized in triplicate to a microarray (‘chip’) containing 477 cDNAs (see Methods for chip design) plus 30 cDNAs encoding plant genes (for the determina- tion of non-specific nucleic acid hybridization). Expression in each neuronal set (called S1, S2 and S3 for small and L1 and L2 for large neurons) was thus monitored in triplicate, requiring a total of 15 microarrays. The quality of the microarray data is demonstrated by pseudocolor arrays, one resulting from hy- bridization to probes derived from neuronal set S1 and the other from neuronal set L1 (Fig. 2a). In Fig. 2a, the enlarged part of the chip shows some differences in fluorescence intensity (that is, expression levels) for particular cDNAs and demonstrates that spots containing the different cDNAs are relatively uniform in size and that background between spots is relatively low. To de- termine whether a signal corresponding to a particular cDNA is reproducible between different chips, we calculated the coeffi- cient of variation [c.v. or (standard deviation/mean) × 100%] for each neuronal set. From these values, the overall average c.v. for all 477 cDNAs per neuronal set was calculated to be: 15.81%, 16.93% and 17.75% for S1, S2 and S3, respectively, and 20.17% and 19.55% for L1 and L2, respectively. Independent amplifications (about 10 6 -fold) of different sets of the same neuronal subtype yielded quite similar expression pat- terns. For example, the correlation of signal intensities between S1 and S2 was R 2 = 0.9688, and between S1 and S3 was R 2 = 0.9399 (Fig. 2b). Similar results were obtained for the two sets of Gene expression profiles of laser-captured adjacent neuronal subtypes LIN LUO 1 , RANELLE C. SALUNGA 1 , HONGQING GUO 1 , ANTON BITTNER 1 , K.C. JOY 1 , JOSE E. GALINDO 1 , HUINIAN XIAO 1 , KATHRYN E. ROGERS 2 , JACKSON S. WAN 1 , MICHAEL R. JACKSON 1 & MARK G. ERLANDER 1 1 R.W . Johnson Pharmaceutical Research Institute, 3535 General Atomics Court, Suite 100, San Diego, California 92121, USA 2 R.W . Johnson Pharmaceutical Research Institute, Spring House, Pennsylvania 19477, USA Correspondence should be addressed to M.G.E.; email: merlande@prius.jnj.com Fig. 1 Laser capture microdissection from Nissl-stained sections (10 μm in thickness) of adult rat large and small DRG neurons. Red arrows indi- cate DRG neurons to be captured (top panels). The middle and bottom panels show successful capture and film transfer, respectively. Scale bar represents 200 μm. © 1999 Nature America Inc. • http://medicine.nature.com © 1999 Nature America Inc. • http://medicine.nature.com