A formalin-free method for stabilizing cells for nucleic acid amplification, hybridization and next-generation sequencing
| Autor: | Jianbing Qin, Erin E. Kaspar, Pamela A. Althof, Bradford A. Hunsley, Jeff Kittrell, Jennifer N. Sanmann |
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| Jazyk: | angličtina |
| Předmět: |
Tissue Fixation
Cell In situ hybridization Biology General Biochemistry Genetics and Molecular Biology law.invention FISH law Cell Line Tumor medicine Technical Note Humans Nucleic acid amplification Polymerase chain reaction Formalin-free fixative In Situ Hybridization Fluorescence Medicine(all) medicine.diagnostic_test Biochemistry Genetics and Molecular Biology(all) Cell fixation General Medicine Nucleic acid amplification technique Sequence Analysis DNA Molecular biology DNA extraction medicine.anatomical_structure Cell culture NGS Nucleic acid Nucleic Acid Amplification Techniques Fluorescence in situ hybridization |
| Zdroj: | BMC Research Notes |
| ISSN: | 1756-0500 |
| DOI: | 10.1186/s13104-015-1725-4 |
| Popis: | Background Formalin has been widely used by pathology laboratories. Its carcinogenicity has led researchers to explore formalin substitutes. Streck Cell Preservative (SCP) is a formalin-free preservative that can preserve cellular antigens. This study was undertaken to investigate the effects of cell preservation using SCP on nucleic acid amplification, hybridization, and next-generation sequencing (NGS) as compared to control frozen cells and cells fixed in the traditional cell and tissue fixative, 10 % neutral buffered formalin (NBF). Findings The breast cancer cell line, SKBR-3, was used as a model system. Prior to nucleic acid extraction and fluorescence in situ hybridization (FISH), cells were fixed in SCP or NBF overnight at room temperature with frozen cells in parallel. Analysis showed that similar DNA extraction yields and amplification profiles determined by PCR in SCP preserved cells and control frozen cells, whereas NBF preserved cells had decreased DNA yield and impaired PCR amplification. Molecular cytogenetic studies by FISH technique indicated that the ratios of ERBB2 (HER-2/neu) signals to the chromosome 17 centromere (CEP17) were comparable for frozen cells and SCP preserved cells. The fluorescence images of both SCP fixed and control frozen cells were also clear and comparable. On the contrary, the same analysis was unsuccessful with NBF preserved cells due to poor hybridization quality. Our data also demonstrated that SCP had negligible effect on NGS testing. Conclusion We conclude that SCP can be used as an alternative to NBF as a preservative for maintaining the integrity of nucleic acids for nucleic acid amplification, sequencing and FISH analysis. Electronic supplementary material The online version of this article (doi:10.1186/s13104-015-1725-4) contains supplementary material, which is available to authorized users. |
| Databáze: | OpenAIRE |
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