Protocol Improvement for RNA Extraction From Compromised Frozen Specimens Generated in Austere Conditions: A Path Forward to Transcriptomics-Pathology Systems Integration
At the heart of the phenome-to-genome approach is high throughput assays, which are liable to produce false results. This risk can be mitigated by minimizing the sample bias, specifically, recycling the same tissue specimen for both phenotypic and genotypic investigations. Therefore, our aim is to s...
| Main Authors: | , , , , , , , |
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| Format: | Article |
| Language: | English |
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Frontiers Media S.A.
2020-07-01
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| Series: | Frontiers in Molecular Biosciences |
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| Online Access: | https://www.frontiersin.org/article/10.3389/fmolb.2020.00142/full |
| _version_ | 1818996304616882176 |
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| author | Nabarun Chakraborty Nabarun Chakraborty Connie W. Schmitt Cary L. Honnold Candace Moyler Candace Moyler Stephen Butler Stephen Butler Hisham Nachabe Hisham Nachabe Aarti Gautam Rasha Hammamieh |
| author_facet | Nabarun Chakraborty Nabarun Chakraborty Connie W. Schmitt Cary L. Honnold Candace Moyler Candace Moyler Stephen Butler Stephen Butler Hisham Nachabe Hisham Nachabe Aarti Gautam Rasha Hammamieh |
| author_sort | Nabarun Chakraborty |
| collection | DOAJ |
| description | At the heart of the phenome-to-genome approach is high throughput assays, which are liable to produce false results. This risk can be mitigated by minimizing the sample bias, specifically, recycling the same tissue specimen for both phenotypic and genotypic investigations. Therefore, our aim is to suggest a methodology of obtaining robust results from frozen specimens of compromised quality, particularly if the sample is produced in conditions with limited resources. For example, generating samples at the International Space Station (ISS) is challenging because the time and laboratory footprint allotted to a project can get expensive. In an effort to be economical with available resources, snap-frozen euthanized mice are the straightforward solution; however, this method increases the risk of temperature abuse during the thawing process at the beginning of the tissue collection. We found that prolonged immersion of snap frozen mouse carcass in 10% neutral buffered formalin at 4°C yielded minimal microscopic signs of ice crystallization and delivered tissues with histomorphology that is optimal for hematoxylin and eosin (H&E) staining and fixation on glass slides. We further optimized a method to sequester the tissue specimen from the H&E slides using an incubator shaker. Using this method, we were able to recover an optimal amount of RNA that could be used for downstream transcriptomics assays. Overall, we demonstrated a protocol that enables us to maximize scientific values from tissues collected in austere condition. Furthermore, our protocol can suggest an improvement in the spatial resolution of transcriptomic assays. |
| first_indexed | 2024-12-20T21:27:37Z |
| format | Article |
| id | doaj.art-c426c07b82ee4fae9ddf8527b8a7ecda |
| institution | Directory Open Access Journal |
| issn | 2296-889X |
| language | English |
| last_indexed | 2024-12-20T21:27:37Z |
| publishDate | 2020-07-01 |
| publisher | Frontiers Media S.A. |
| record_format | Article |
| series | Frontiers in Molecular Biosciences |
| spelling | doaj.art-c426c07b82ee4fae9ddf8527b8a7ecda2022-12-21T19:26:07ZengFrontiers Media S.A.Frontiers in Molecular Biosciences2296-889X2020-07-01710.3389/fmolb.2020.00142539955Protocol Improvement for RNA Extraction From Compromised Frozen Specimens Generated in Austere Conditions: A Path Forward to Transcriptomics-Pathology Systems IntegrationNabarun Chakraborty0Nabarun Chakraborty1Connie W. Schmitt2Cary L. Honnold3Candace Moyler4Candace Moyler5Stephen Butler6Stephen Butler7Hisham Nachabe8Hisham Nachabe9Aarti Gautam10Rasha Hammamieh11Geneva Foundation, Walter Reed Army Institute of Research, Silver Spring, MD, United StatesMedical Readiness Systems Biology, Walter Reed Army Institute of Research, Silver Spring, MD, United StatesComparative Pathology, US Army Medical Research Institute of Chemical Defense, Gunpowder, MD, United StatesComparative Pathology, US Army Medical Research Institute of Chemical Defense, Gunpowder, MD, United StatesMedical Readiness Systems Biology, Walter Reed Army Institute of Research, Silver Spring, MD, United StatesORISE, Walter Reed Army Institute of Research, Silver Spring, MD, United StatesGeneva Foundation, Walter Reed Army Institute of Research, Silver Spring, MD, United StatesMedical Readiness Systems Biology, Walter Reed Army Institute of Research, Silver Spring, MD, United StatesMedical Readiness Systems Biology, Walter Reed Army Institute of Research, Silver Spring, MD, United StatesORISE, Walter Reed Army Institute of Research, Silver Spring, MD, United StatesMedical Readiness Systems Biology, Walter Reed Army Institute of Research, Silver Spring, MD, United StatesMedical Readiness Systems Biology, Walter Reed Army Institute of Research, Silver Spring, MD, United StatesAt the heart of the phenome-to-genome approach is high throughput assays, which are liable to produce false results. This risk can be mitigated by minimizing the sample bias, specifically, recycling the same tissue specimen for both phenotypic and genotypic investigations. Therefore, our aim is to suggest a methodology of obtaining robust results from frozen specimens of compromised quality, particularly if the sample is produced in conditions with limited resources. For example, generating samples at the International Space Station (ISS) is challenging because the time and laboratory footprint allotted to a project can get expensive. In an effort to be economical with available resources, snap-frozen euthanized mice are the straightforward solution; however, this method increases the risk of temperature abuse during the thawing process at the beginning of the tissue collection. We found that prolonged immersion of snap frozen mouse carcass in 10% neutral buffered formalin at 4°C yielded minimal microscopic signs of ice crystallization and delivered tissues with histomorphology that is optimal for hematoxylin and eosin (H&E) staining and fixation on glass slides. We further optimized a method to sequester the tissue specimen from the H&E slides using an incubator shaker. Using this method, we were able to recover an optimal amount of RNA that could be used for downstream transcriptomics assays. Overall, we demonstrated a protocol that enables us to maximize scientific values from tissues collected in austere condition. Furthermore, our protocol can suggest an improvement in the spatial resolution of transcriptomic assays.https://www.frontiersin.org/article/10.3389/fmolb.2020.00142/fullassay optimizationhistopatholgytranscriptomicsproduct developmentsystems integration |
| spellingShingle | Nabarun Chakraborty Nabarun Chakraborty Connie W. Schmitt Cary L. Honnold Candace Moyler Candace Moyler Stephen Butler Stephen Butler Hisham Nachabe Hisham Nachabe Aarti Gautam Rasha Hammamieh Protocol Improvement for RNA Extraction From Compromised Frozen Specimens Generated in Austere Conditions: A Path Forward to Transcriptomics-Pathology Systems Integration Frontiers in Molecular Biosciences assay optimization histopatholgy transcriptomics product development systems integration |
| title | Protocol Improvement for RNA Extraction From Compromised Frozen Specimens Generated in Austere Conditions: A Path Forward to Transcriptomics-Pathology Systems Integration |
| title_full | Protocol Improvement for RNA Extraction From Compromised Frozen Specimens Generated in Austere Conditions: A Path Forward to Transcriptomics-Pathology Systems Integration |
| title_fullStr | Protocol Improvement for RNA Extraction From Compromised Frozen Specimens Generated in Austere Conditions: A Path Forward to Transcriptomics-Pathology Systems Integration |
| title_full_unstemmed | Protocol Improvement for RNA Extraction From Compromised Frozen Specimens Generated in Austere Conditions: A Path Forward to Transcriptomics-Pathology Systems Integration |
| title_short | Protocol Improvement for RNA Extraction From Compromised Frozen Specimens Generated in Austere Conditions: A Path Forward to Transcriptomics-Pathology Systems Integration |
| title_sort | protocol improvement for rna extraction from compromised frozen specimens generated in austere conditions a path forward to transcriptomics pathology systems integration |
| topic | assay optimization histopatholgy transcriptomics product development systems integration |
| url | https://www.frontiersin.org/article/10.3389/fmolb.2020.00142/full |
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