Optimizing Laser Capture Microdissection Protocol for Isolating Zone-Specific Cell Populations from Mandibular Condylar Cartilage
Mandibular condylar cartilage (MCC) is a multizonal heterogeneous fibrocartilage consisting of fibrous (FZ), proliferative (PZ), mature (MZ), and hypertrophic (HZ) zones. Gross sampling of the whole tissue may conceal some important information and compromise the validity of the molecular analysis....
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Wiley
2019-01-01
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| Series: | International Journal of Dentistry |
| Online Access: | http://dx.doi.org/10.1155/2019/5427326 |
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| author | Aisha M. Basudan Yanqi Yang |
| author_facet | Aisha M. Basudan Yanqi Yang |
| author_sort | Aisha M. Basudan |
| collection | DOAJ |
| description | Mandibular condylar cartilage (MCC) is a multizonal heterogeneous fibrocartilage consisting of fibrous (FZ), proliferative (PZ), mature (MZ), and hypertrophic (HZ) zones. Gross sampling of the whole tissue may conceal some important information and compromise the validity of the molecular analysis. Laser capture microdissection (LCM) technology allows isolating zonal (homogenous) cell populations and consequently generating more accurate molecular and genetic data, but the challenges during tissue preparation and microdissection procedures are to obtain acceptable tissue section morphology that allows histological identification of the desirable cell type and to minimize RNA degradation. Therefore, our aim is to optimize an LCM protocol for isolating four homogenous zone-specific cell populations from their respective MCC zones while preserving the quality of RNA recovered. MCC and FCC (femoral condylar cartilage) specimens were harvested from 5-week-old Sprague–Dawley male rats. Formalin-fixed and frozen unfixed tissue sections were prepared and compared histologically. Additional specimens were microdissected to prepare LCM samples from FCC and each MCC zone individually. Then, to evaluate LCM-RNA integrity, 3′/m ratios of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and beta-actin (β-Actin) using quantitative reverse transcription-polymerase chain reaction (qRT-PCR) were calculated. Both fixed and unfixed tissue sections allowed reliable identification of MCC zones. The improved morphology of the frozen sections of our protocol has extended the range of cell types to be isolated. Under the empirically set LCM parameters, four homogeneous cell populations were efficiently isolated from their respective zones. The 3′/m ratio means of GAPDH and β-Actin ranged between 1.11–1.56 and 1.41–2.12, respectively. These values are in line with the reported quality control requirements. The present study shows that the optimized LCM protocol could allow isolation of four homogenous zone-specific cell populations from MCC, meanwhile preserving RNA integrity to meet the high quality requirements for subsequent molecular analyses. Thereby, accurate molecular and genetic data could be generated. |
| format | Article |
| id | doaj-art-e35cbfd828e84a4992c85fd0e369b624 |
| institution | Kabale University |
| issn | 1687-8728 1687-8736 |
| language | English |
| publishDate | 2019-01-01 |
| publisher | Wiley |
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| series | International Journal of Dentistry |
| spelling | doaj-art-e35cbfd828e84a4992c85fd0e369b6242025-02-03T01:23:48ZengWileyInternational Journal of Dentistry1687-87281687-87362019-01-01201910.1155/2019/54273265427326Optimizing Laser Capture Microdissection Protocol for Isolating Zone-Specific Cell Populations from Mandibular Condylar CartilageAisha M. Basudan0Yanqi Yang1Division of Orthodontics, Dental Services Department, Ministry of National Guard-Health Affairs, Riyadh, Saudi ArabiaDepartment of Orthodontics, Faculty of Dentistry, The University of Hong Kong, Pokfulam, Hong KongMandibular condylar cartilage (MCC) is a multizonal heterogeneous fibrocartilage consisting of fibrous (FZ), proliferative (PZ), mature (MZ), and hypertrophic (HZ) zones. Gross sampling of the whole tissue may conceal some important information and compromise the validity of the molecular analysis. Laser capture microdissection (LCM) technology allows isolating zonal (homogenous) cell populations and consequently generating more accurate molecular and genetic data, but the challenges during tissue preparation and microdissection procedures are to obtain acceptable tissue section morphology that allows histological identification of the desirable cell type and to minimize RNA degradation. Therefore, our aim is to optimize an LCM protocol for isolating four homogenous zone-specific cell populations from their respective MCC zones while preserving the quality of RNA recovered. MCC and FCC (femoral condylar cartilage) specimens were harvested from 5-week-old Sprague–Dawley male rats. Formalin-fixed and frozen unfixed tissue sections were prepared and compared histologically. Additional specimens were microdissected to prepare LCM samples from FCC and each MCC zone individually. Then, to evaluate LCM-RNA integrity, 3′/m ratios of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and beta-actin (β-Actin) using quantitative reverse transcription-polymerase chain reaction (qRT-PCR) were calculated. Both fixed and unfixed tissue sections allowed reliable identification of MCC zones. The improved morphology of the frozen sections of our protocol has extended the range of cell types to be isolated. Under the empirically set LCM parameters, four homogeneous cell populations were efficiently isolated from their respective zones. The 3′/m ratio means of GAPDH and β-Actin ranged between 1.11–1.56 and 1.41–2.12, respectively. These values are in line with the reported quality control requirements. The present study shows that the optimized LCM protocol could allow isolation of four homogenous zone-specific cell populations from MCC, meanwhile preserving RNA integrity to meet the high quality requirements for subsequent molecular analyses. Thereby, accurate molecular and genetic data could be generated.http://dx.doi.org/10.1155/2019/5427326 |
| spellingShingle | Aisha M. Basudan Yanqi Yang Optimizing Laser Capture Microdissection Protocol for Isolating Zone-Specific Cell Populations from Mandibular Condylar Cartilage International Journal of Dentistry |
| title | Optimizing Laser Capture Microdissection Protocol for Isolating Zone-Specific Cell Populations from Mandibular Condylar Cartilage |
| title_full | Optimizing Laser Capture Microdissection Protocol for Isolating Zone-Specific Cell Populations from Mandibular Condylar Cartilage |
| title_fullStr | Optimizing Laser Capture Microdissection Protocol for Isolating Zone-Specific Cell Populations from Mandibular Condylar Cartilage |
| title_full_unstemmed | Optimizing Laser Capture Microdissection Protocol for Isolating Zone-Specific Cell Populations from Mandibular Condylar Cartilage |
| title_short | Optimizing Laser Capture Microdissection Protocol for Isolating Zone-Specific Cell Populations from Mandibular Condylar Cartilage |
| title_sort | optimizing laser capture microdissection protocol for isolating zone specific cell populations from mandibular condylar cartilage |
| url | http://dx.doi.org/10.1155/2019/5427326 |
| work_keys_str_mv | AT aishambasudan optimizinglasercapturemicrodissectionprotocolforisolatingzonespecificcellpopulationsfrommandibularcondylarcartilage AT yanqiyang optimizinglasercapturemicrodissectionprotocolforisolatingzonespecificcellpopulationsfrommandibularcondylarcartilage |