Analysis of connective tissues by laser capture microdissection and reverse transcriptase-polymerase chain reaction.
Studies of gene expression from bone, cartilage, and other tissues are complicated by the fact that their RNA, collected and pooled for analysis, often represents a wide variety of composite cells distinct in individual phenotype, age, and state of maturation. Laser capture microdissection (LCM) is a technique that allows specific cells to be isolated according to their phenotype, condition, or other marker from within such heterogeneity. As a result, this approach can yield RNA that is particular to a subset of cells comprising the total cell population of the tissue. This study reports the application of LCM to the gene expression analysis of the cartilaginous epiphyseal growth plate of normal newborn mice. The methodology utilized for this purpose has been coupled with real-time quantitative reverse transcriptase-polymerase chain reaction (QRT-PCR) to quantitate the expression of certain genes involved in growth plate development and calcification. In this paper, the approaches used for isolating and purifying RNA from phenotypically specific chondrocyte populations of the murine growth plate are detailed and illustrate and compare both qualitative and quantitative RT-PCR results. The technique will hopefully serve as a guide for the further analysis of this and other connective tissues by LCM and RT-PCR.
Jacquet
Robin
R
Department of Biochemistry and Molecular Pathology, Northeastern Ohio Universities College of Medicine, Rootstown, OH 44272, USA.
Hillyer
Jennifer
J
Landis
William J
WJ
D000818:Animals; D000831:Animals, Newborn; D002469:Cell Separation / Q000379:methods*; D019902:Chondrocytes / Q000737:chemistry / Q000166:cytology; D003239:Connective Tissue Cells / Q000737:chemistry / Q000166:cytology*; D020869:Gene Expression Profiling / Q000379:methods*; D006132:Growth Plate / Q000166:cytology; D007834:Lasers; D008722:Methods; D051379:Mice; D008815:Mice, Inbred Strains; D042282:Microdissection; D012333:RNA, Messenger / Q000032:analysis / Q000302:isolation & purification*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013977:Tibia / Q000166:cytology
D012333:RNA, Messenger