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Laser-capture microdissection of murine lung for differential cellular RNA analysis

  • Jagadish Loganathan
  • Roshni Pandey
  • Nilesh Sudhakar Ambhore
  • Pawel Borowicz
  • Venkatachalem SathishEmail author
Regular Article
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Abstract

The lung tissue contains a heterogeneous milieu of bronchioles, epithelial, airway smooth muscle (ASM), alveolar, and immune cell types. Healthy bronchiole comprises epithelial cells surrounded by ASM cells and helps in normal respiration. In contrast, airway remodeling, or plasticity, increases surrounding of bronchial epithelium during inflammation, especially in asthmatic condition. Given the profound functional difference between ASM, epithelial, and other cell types in the lung, it is imperative to separate and isolate different cell types of lungs for genomics, proteomics, and molecular analysis, which will improve the diagnostic and therapeutic approach to treat cell-specific lung disorders. Laser capture microdissection (LCM) is the technique generally used for the isolation of specific cell populations under direct visual inspection, which plays a crucial role to evaluate cell-specific effect in clinical and preclinical setup. However, maintenance of tissue RNA quality and integrity in LCM studies are very challenging tasks. It is obvious to believe that the major factor affecting the RNA quality is tissue-fixation method. The prime focus of this study was to address the RNA quality factors within the lung tissue using the different solvent system to fix tissue sample to obtain high-quality RNA. Paraformaldehyde and Carnoy’s solutions were used for fixing the lung tissue and compared RNA integrity in LCM captured lung tissue samples. To further confirm the quality of RNA, we measured cellular marker genes in collected lung tissue samples from control and mixed allergen (MA)-induced asthmatic mouse model using qRT-PCR technique. RNA integrity number showed a significantly better quality of RNA in lung tissue samples fixed with Carnoy’s solution compared to paraformaldehyde solution. Isolated RNA from MA-induced asthmatic murine lung epithelium, smooth muscle, and granulomatous foci using LCM showed a significant increase in remodeling gene expression compared to control which confirm the quality and integrity of isolated RNA. Overall, the study concludes tissue fixation solvent can alter the quality of RNA in the lung and the outcome of the results.

Keywords

Carnoy’s solution RNA integrity Lung tissue fixation Laser capture microdissection qRT-PCR 

Notes

Author contributions

JL, RP, NSA, and SV have conceptualized the study. JL, RP, and NSA have performed the experimental procedures. JL, RP, NSA, and SV have contributed in data analysis and manuscript preparation. SV revised final draft and approved for submission.

Funding information

Supported by NIH grants R01 HL123494 (Venkatachalem). Additional support in part from ND EPSCoR with NSF #1355466 and NDSU RCA Activity.

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Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  • Jagadish Loganathan
    • 1
  • Roshni Pandey
    • 1
  • Nilesh Sudhakar Ambhore
    • 1
  • Pawel Borowicz
    • 2
  • Venkatachalem Sathish
    • 1
    Email author
  1. 1.Department of Pharmaceutical Sciences, School of PharmacyNorth Dakota State UniversityFargoUSA
  2. 2.Department of Animal SciencesNorth Dakota State UniversityFargoUSA

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