Gene expression analysis of distinct populations of cells isolated from mouse and human inner ear FFPE tissue using laser capture microdissection--a technical report based on preliminary findings.
Laser Capture Microdissection (LCM) allows microscopic procurement of specific cell types from tissue sections that can then be used for gene expression analysis. We first tested this method with sections of adult mouse inner ears and subsequently applied it to human inner ear sections. The morphology of the various cell types within the inner ear is well preserved in formalin fixed paraffin embedded (FFPE) sections, making it easier to identify cell types and their boundaries. Recovery of good quality RNA from FFPE sections can be challenging, however, recent studies in cancer research demonstrated that it is possible to carry out gene expression analysis of FFPE material. Thus, a method developed using mouse FFPE tissue can be applied to human archival temporal bones. This is important because the majority of human temporal bone banks have specimens preserved in formalin and a technique for retrospective analysis of human archival ear tissue is needed. We used mouse FFPE inner ear sections to procure distinct populations of cells from the various functional domains (organ of Corti, spiral ganglion, etc.) by LCM. RNA was extracted from captured cells, amplified, and assessed for quality. Expression of selected genes was tested by RT-PCR. In addition to housekeeping genes, we were able to detect cell type specific markers, such as Myosin 7a, p27(kip1) and neurofilament gene transcripts that confirmed the likely composition of cells in the sample. We also tested the method described above on FFPE sections from human crista ampullaris. These sections were approximately a year old. Populations of cells from the epithelium and stroma were collected and analyzed independently for gene expression. The method described here has potential use in many areas of hearing research. For example, following exposure to noise, ototoxic drugs or age, it would be highly desirable to analyze gene expression profiles of selected populations of cells within the organ of Corti or spiral ganglion cells rather than a mixed population of cells from whole inner ear tissue. Also, this method can be applied for analysis of human archival ear tissue.
Pagedar
Nitin A
NA
Department of Otolaryngology-Head and Neck Surgery, University Hospitals of Cleveland, Lakeside 4500, 11100 Euclid Avenue, Cleveland, OH 44106, USA.
Wang
Wen
W
Chen
Daniel H-C
DH
Davis
Rickie R
RR
Lopez
Ivan
I
Wright
Charles G
CG
Alagramam
Kumar N
KN
D000818:Animals; D002452:Cell Count / Q000379:methods; D050760:Cyclin-Dependent Kinase Inhibitor p27 / Q000235:genetics / Q000378:metabolism; D004398:Dyneins / Q000235:genetics / Q000378:metabolism; D007758:Ear, Inner / Q000033:anatomy & histology / Q000378:metabolism*; D015870:Gene Expression / Q000502:physiology*; D006801:Humans; D017403:In Situ Hybridization / Q000379:methods; D007834:Lasers; D051379:Mice; D042282:Microdissection / Q000379:methods*; D000081412:Myosin VIIa; D009218:Myosins / Q000235:genetics / Q000378:metabolism; D016900:Neurofilament Proteins / Q000235:genetics / Q000378:metabolism; D016612:Paraffin Embedding / Q000379:methods*; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D016707:Tissue Fixation / Q000379:methods
C000631455:MYO7A protein, human; C000631456:Myo7a protein, mouse; D000081412:Myosin VIIa; D016900:Neurofilament Proteins; D012333:RNA, Messenger; C051774:neurofilament protein H; D050760:Cyclin-Dependent Kinase Inhibitor p27; D009218:Myosins; D004398:Dyneins