cDNA array hybridization after laser-assisted microdissection from nonneoplastic tissue.
Differential gene expression can be investigated effectively by cDNA arrays. Because tissue homogenates result inevitably in an average expression of a bulk of different cells, we aimed to combine mRNA profiling with cell-type-specific microdissection. Using a polymerase chain reaction (PCR)-based preamplification technique, the expression profile was shown to be preserved. We modified the existing protocol enabling to apply the total amount of extracted RNA from microdissected cells. A mean amplification factor of nearly 1000 allowed to reduce the demand of initial RNA to approximately 10 ng. This technique was used to investigate intrapulmonary arteries from mouse lungs ( approximately 500 cell equivalents). Using filters with 1176 spots, three independent experiments showed a high consistency of expression for the preamplified cDNAs. These profiles differed primarily from those of total lung homogenates. Additionally, in experimental hypoxia-induced pulmonary hypertension, amplified cDNA from intrapulmonary vessels of these lungs was compared to cDNA from vessels dissected from normoxic lungs. Validation by an alternative method was obtained by linking microdissection with real-time polymerase chain reaction (PCR). As suggested by the array data, nine selected genes with different factors of up-regulation were fully confirmed by the PCR technique. Thus, a rapid protocol is presented combining microdissection and array profiling that demands low quantities of initial RNA to assess reliably cell-type-specific gene regulation even within nonneoplastic complex tissues.
Fink
Ludger
L
Department of Pathology, Justus-Liebig-University Giessen, Giessen, Germany. ludger.fink@patho.med.uni-giessen.de
Kohlhoff
Stephanie
S
Stein
Maria Magdalena
MM
Hänze
Jörg
J
Weissmann
Norbert
N
Rose
Frank
F
Akkayagil
Ercan
E
Manz
Daniel
D
Grimminger
Friedrich
F
Seeger
Werner
W
Bohle
Rainer Maria
RM
D000818:Animals; D003199:Computer Systems; D018076:DNA, Complementary / Q000235:genetics; D004210:Dissection / Q000379:methods; D020869:Gene Expression Profiling; D000860:Hypoxia / Q000235:genetics; D007834:Lasers; D008168:Lung / Q000473:pathology / Q000503:physiopathology; D051379:Mice; D008807:Mice, Inbred BALB C; D020411:Oligonucleotide Array Sequence Analysis; D016133:Polymerase Chain Reaction; D011651:Pulmonary Artery / Q000166:cytology / Q000502:physiology*; D012333:RNA, Messenger / Q000378:metabolism; D012016:Reference Values; D015203:Reproducibility of Results
D018076:DNA, Complementary; D012333:RNA, Messenger