Comparative evaluation of ACetic - MEthanol high salt dissociation approach for single-cell transcriptomics of frozen human tissues
Current dissociation methods for solid tissues in scRNA-seq studies do not guarantee intact single-cell isolation, especially for sensitive and complex human endocrine tissues. Most studies rely on enzymatic dissociation of fresh samples or nuclei isolation from frozen samples. Dissociating whole in...
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Frontiers Media S.A.
2025-01-01
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| author | Marina Utkina Anastasia Shcherbakova Ruslan Deviatiiarov Ruslan Deviatiiarov Ruslan Deviatiiarov Alina Ryabova Marina Loguinova Valentin Trofimov Anna Kuznetsova Mikhail Petropavlovskiy Rustam Salimkhanov Denis Maksimov Eugene Albert Alexandra Golubeva Walaa Asaad Lilia Urusova Ekaterina Bondarenko Anastasia Lapshina Alexandra Shutova Dmitry Beltsevich Oleg Gusev Oleg Gusev Oleg Gusev Oleg Gusev Larisa Dzeranova Galina Melnichenko Ildar Minniakhmetov Ivan Dedov Natalya Mokrysheva Sergey Popov |
| author_facet | Marina Utkina Anastasia Shcherbakova Ruslan Deviatiiarov Ruslan Deviatiiarov Ruslan Deviatiiarov Alina Ryabova Marina Loguinova Valentin Trofimov Anna Kuznetsova Mikhail Petropavlovskiy Rustam Salimkhanov Denis Maksimov Eugene Albert Alexandra Golubeva Walaa Asaad Lilia Urusova Ekaterina Bondarenko Anastasia Lapshina Alexandra Shutova Dmitry Beltsevich Oleg Gusev Oleg Gusev Oleg Gusev Oleg Gusev Larisa Dzeranova Galina Melnichenko Ildar Minniakhmetov Ivan Dedov Natalya Mokrysheva Sergey Popov |
| author_sort | Marina Utkina |
| collection | DOAJ |
| description | Current dissociation methods for solid tissues in scRNA-seq studies do not guarantee intact single-cell isolation, especially for sensitive and complex human endocrine tissues. Most studies rely on enzymatic dissociation of fresh samples or nuclei isolation from frozen samples. Dissociating whole intact cells from fresh-frozen samples, commonly collected by biobanks, remains a challenge. Here, we utilized the acetic-methanol dissociation approach (ACME) to capture transcriptional profiles of individual cells from fresh-frozen tissue samples. This method combines acetic acid-based dissociation and methanol-based fixation. In our study, we optimized this approach for human endocrine tissue samples for the first time. We incorporated a high-salt washing buffer instead of the standard PBS to stabilize RNA and prevent RNases reactivation during rehydration. We have designated this optimized protocol as ACME HS (ACetic acid-MEthanol High Salt). This technique aims to preserve cell morphology and RNA integrity, minimizing transcriptome changes and providing a more accurate representation of mature mRNA. We compared the ability of enzymatic, ACME HS, and nuclei isolation methods to preserve major cell types, gene expression, and standard quality parameters across 41 tissue samples. Our results demonstrated that ACME HS effectively dissociates and fixes cells, preserving cell morphology and high RNA integrity. This makes ACME HS a valuable alternative for scRNA-seq protocols involving challenging tissues where obtaining a live cell suspension is difficult or disruptive. |
| format | Article |
| id | doaj-art-fb93e43d46294ca99128e8fda7c24f74 |
| institution | Kabale University |
| issn | 2296-634X |
| language | English |
| publishDate | 2025-01-01 |
| publisher | Frontiers Media S.A. |
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| series | Frontiers in Cell and Developmental Biology |
| spelling | doaj-art-fb93e43d46294ca99128e8fda7c24f742025-01-07T13:01:23ZengFrontiers Media S.A.Frontiers in Cell and Developmental Biology2296-634X2025-01-011210.3389/fcell.2024.14699551469955Comparative evaluation of ACetic - MEthanol high salt dissociation approach for single-cell transcriptomics of frozen human tissuesMarina Utkina0Anastasia Shcherbakova1Ruslan Deviatiiarov2Ruslan Deviatiiarov3Ruslan Deviatiiarov4Alina Ryabova5Marina Loguinova6Valentin Trofimov7Anna Kuznetsova8Mikhail Petropavlovskiy9Rustam Salimkhanov10Denis Maksimov11Eugene Albert12Alexandra Golubeva13Walaa Asaad14Lilia Urusova15Ekaterina Bondarenko16Anastasia Lapshina17Alexandra Shutova18Dmitry Beltsevich19Oleg Gusev20Oleg Gusev21Oleg Gusev22Oleg Gusev23Larisa Dzeranova24Galina Melnichenko25Ildar Minniakhmetov26Ivan Dedov27Natalya Mokrysheva28Sergey Popov29Endocrinology Research Centre, Institute of Personalized Medicine, Moscow, RussiaEndocrinology Research Centre, Institute of Personalized Medicine, Moscow, RussiaEndocrinology Research Centre, Institute of Personalized Medicine, Moscow, RussiaGraduate School of Medicine, Juntendo University, Bunkyo-ku, JapanLife Improvement by Future Technologies (LIFT) Center, Moscow, RussiaEndocrinology Research Centre, Institute of Personalized Medicine, Moscow, RussiaEndocrinology Research Centre, Institute of Personalized Medicine, Moscow, RussiaEndocrinology Research Centre, Institute of Personalized Medicine, Moscow, RussiaEndocrinology Research Centre, Institute of Personalized Medicine, Moscow, RussiaEndocrinology Research Centre, Institute of Personalized Medicine, Moscow, RussiaEndocrinology Research Centre, Institute of Personalized Medicine, Moscow, RussiaFaculty of Medicine, Lomonosov Moscow State University, Moscow, RussiaFaculty of Medicine, Lomonosov Moscow State University, Moscow, RussiaEndocrinology Research Centre, Institute of Personalized Medicine, Moscow, RussiaEndocrinology Research Centre, Institute of Personalized Medicine, Moscow, RussiaEndocrinology Research Centre, Institute of Personalized Medicine, Moscow, RussiaEndocrinology Research Centre, Institute of Personalized Medicine, Moscow, RussiaEndocrinology Research Centre, Institute of Personalized Medicine, Moscow, RussiaEndocrinology Research Centre, Institute of Personalized Medicine, Moscow, RussiaEndocrinology Research Centre, Institute of Personalized Medicine, Moscow, RussiaEndocrinology Research Centre, Institute of Personalized Medicine, Moscow, RussiaGraduate School of Medicine, Juntendo University, Bunkyo-ku, JapanLife Improvement by Future Technologies (LIFT) Center, Moscow, RussiaRegulatory Genomics Research Center, Institute of Fundamental Medicine and Biology, Kazan Federal University, Kazan, RussiaEndocrinology Research Centre, Institute of Personalized Medicine, Moscow, RussiaEndocrinology Research Centre, Institute of Personalized Medicine, Moscow, RussiaEndocrinology Research Centre, Institute of Personalized Medicine, Moscow, RussiaEndocrinology Research Centre, Institute of Personalized Medicine, Moscow, RussiaEndocrinology Research Centre, Institute of Personalized Medicine, Moscow, RussiaEndocrinology Research Centre, Institute of Personalized Medicine, Moscow, RussiaCurrent dissociation methods for solid tissues in scRNA-seq studies do not guarantee intact single-cell isolation, especially for sensitive and complex human endocrine tissues. Most studies rely on enzymatic dissociation of fresh samples or nuclei isolation from frozen samples. Dissociating whole intact cells from fresh-frozen samples, commonly collected by biobanks, remains a challenge. Here, we utilized the acetic-methanol dissociation approach (ACME) to capture transcriptional profiles of individual cells from fresh-frozen tissue samples. This method combines acetic acid-based dissociation and methanol-based fixation. In our study, we optimized this approach for human endocrine tissue samples for the first time. We incorporated a high-salt washing buffer instead of the standard PBS to stabilize RNA and prevent RNases reactivation during rehydration. We have designated this optimized protocol as ACME HS (ACetic acid-MEthanol High Salt). This technique aims to preserve cell morphology and RNA integrity, minimizing transcriptome changes and providing a more accurate representation of mature mRNA. We compared the ability of enzymatic, ACME HS, and nuclei isolation methods to preserve major cell types, gene expression, and standard quality parameters across 41 tissue samples. Our results demonstrated that ACME HS effectively dissociates and fixes cells, preserving cell morphology and high RNA integrity. This makes ACME HS a valuable alternative for scRNA-seq protocols involving challenging tissues where obtaining a live cell suspension is difficult or disruptive.https://www.frontiersin.org/articles/10.3389/fcell.2024.1469955/fullScRNA-seqACME HScryopreservationdissociationfresh-frozen tissuefixed single cells |
| spellingShingle | Marina Utkina Anastasia Shcherbakova Ruslan Deviatiiarov Ruslan Deviatiiarov Ruslan Deviatiiarov Alina Ryabova Marina Loguinova Valentin Trofimov Anna Kuznetsova Mikhail Petropavlovskiy Rustam Salimkhanov Denis Maksimov Eugene Albert Alexandra Golubeva Walaa Asaad Lilia Urusova Ekaterina Bondarenko Anastasia Lapshina Alexandra Shutova Dmitry Beltsevich Oleg Gusev Oleg Gusev Oleg Gusev Oleg Gusev Larisa Dzeranova Galina Melnichenko Ildar Minniakhmetov Ivan Dedov Natalya Mokrysheva Sergey Popov Comparative evaluation of ACetic - MEthanol high salt dissociation approach for single-cell transcriptomics of frozen human tissues Frontiers in Cell and Developmental Biology ScRNA-seq ACME HS cryopreservation dissociation fresh-frozen tissue fixed single cells |
| title | Comparative evaluation of ACetic - MEthanol high salt dissociation approach for single-cell transcriptomics of frozen human tissues |
| title_full | Comparative evaluation of ACetic - MEthanol high salt dissociation approach for single-cell transcriptomics of frozen human tissues |
| title_fullStr | Comparative evaluation of ACetic - MEthanol high salt dissociation approach for single-cell transcriptomics of frozen human tissues |
| title_full_unstemmed | Comparative evaluation of ACetic - MEthanol high salt dissociation approach for single-cell transcriptomics of frozen human tissues |
| title_short | Comparative evaluation of ACetic - MEthanol high salt dissociation approach for single-cell transcriptomics of frozen human tissues |
| title_sort | comparative evaluation of acetic methanol high salt dissociation approach for single cell transcriptomics of frozen human tissues |
| topic | ScRNA-seq ACME HS cryopreservation dissociation fresh-frozen tissue fixed single cells |
| url | https://www.frontiersin.org/articles/10.3389/fcell.2024.1469955/full |
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