A phenol/chloroform-free method to extract nucleic acids from recalcitrant, woody tropical species for gene expression and sequencing
Abstract Background Woody tropical plants contain high levels of complex organic compounds that inhibit the chemical procedures needed to extract RNA or DNA, thus compromising downstream applications such as RNA sequencing and analysis of gene expression. To overcome this issue, researchers must use...
| Main Authors: | , , , , , , |
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| Format: | Article |
| Language: | English |
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BMC
2019-06-01
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| Series: | Plant Methods |
| Subjects: | |
| Online Access: | http://link.springer.com/article/10.1186/s13007-019-0447-3 |
| _version_ | 1819063948171804672 |
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| author | François F. Barbier Tinashe G. Chabikwa Muhammad U. Ahsan Stacey E. Cook Rosanna Powell Milos Tanurdzic Christine A. Beveridge |
| author_facet | François F. Barbier Tinashe G. Chabikwa Muhammad U. Ahsan Stacey E. Cook Rosanna Powell Milos Tanurdzic Christine A. Beveridge |
| author_sort | François F. Barbier |
| collection | DOAJ |
| description | Abstract Background Woody tropical plants contain high levels of complex organic compounds that inhibit the chemical procedures needed to extract RNA or DNA, thus compromising downstream applications such as RNA sequencing and analysis of gene expression. To overcome this issue, researchers must use extraction protocols using CTAB/PVP buffer instead of commercially available DNA/RNA extraction kits. However, these protocols are time-consuming, use toxic chemicals like phenol and chloroform, and can only be used to process a small number of samples at a time. To overcome these issues, we developed a new CTAB/PVP based protocol for RNA or DNA extraction that eliminates the traditional phenol/chloroform step. Furthermore, the protocol was developed for 96-well plates to speed up processing. Results Our new protocol enabled us to successfully extract RNA from macadamia, avocado, and mango tissues that are traditionally difficult to work with. This RNA was then successfully used to synthesise cDNA for real-time quantitative PCR and to generate good quality RNA-Seq libraries. Our protocol can be easily converted for rapid DNA extraction from different tropical and sub-tropical tree species. Conclusion This method enables safer and faster DNA and RNA extraction from recalcitrant species, thus facilitating future work on tropical trees. |
| first_indexed | 2024-12-21T15:22:47Z |
| format | Article |
| id | doaj.art-1df4c63aa28348b3a7b381cd3500869c |
| institution | Directory Open Access Journal |
| issn | 1746-4811 |
| language | English |
| last_indexed | 2024-12-21T15:22:47Z |
| publishDate | 2019-06-01 |
| publisher | BMC |
| record_format | Article |
| series | Plant Methods |
| spelling | doaj.art-1df4c63aa28348b3a7b381cd3500869c2022-12-21T18:58:59ZengBMCPlant Methods1746-48112019-06-0115111310.1186/s13007-019-0447-3A phenol/chloroform-free method to extract nucleic acids from recalcitrant, woody tropical species for gene expression and sequencingFrançois F. Barbier0Tinashe G. Chabikwa1Muhammad U. Ahsan2Stacey E. Cook3Rosanna Powell4Milos Tanurdzic5Christine A. Beveridge6School of Biological Sciences, and The Queensland Alliance for Agriculture and Food Innovation, The University of QueenslandSchool of Biological Sciences, and The Queensland Alliance for Agriculture and Food Innovation, The University of QueenslandSchool of Biological Sciences, and The Queensland Alliance for Agriculture and Food Innovation, The University of QueenslandSchool of Biological Sciences, and The Queensland Alliance for Agriculture and Food Innovation, The University of QueenslandSchool of Biological Sciences, and The Queensland Alliance for Agriculture and Food Innovation, The University of QueenslandSchool of Biological Sciences, and The Queensland Alliance for Agriculture and Food Innovation, The University of QueenslandSchool of Biological Sciences, and The Queensland Alliance for Agriculture and Food Innovation, The University of QueenslandAbstract Background Woody tropical plants contain high levels of complex organic compounds that inhibit the chemical procedures needed to extract RNA or DNA, thus compromising downstream applications such as RNA sequencing and analysis of gene expression. To overcome this issue, researchers must use extraction protocols using CTAB/PVP buffer instead of commercially available DNA/RNA extraction kits. However, these protocols are time-consuming, use toxic chemicals like phenol and chloroform, and can only be used to process a small number of samples at a time. To overcome these issues, we developed a new CTAB/PVP based protocol for RNA or DNA extraction that eliminates the traditional phenol/chloroform step. Furthermore, the protocol was developed for 96-well plates to speed up processing. Results Our new protocol enabled us to successfully extract RNA from macadamia, avocado, and mango tissues that are traditionally difficult to work with. This RNA was then successfully used to synthesise cDNA for real-time quantitative PCR and to generate good quality RNA-Seq libraries. Our protocol can be easily converted for rapid DNA extraction from different tropical and sub-tropical tree species. Conclusion This method enables safer and faster DNA and RNA extraction from recalcitrant species, thus facilitating future work on tropical trees.http://link.springer.com/article/10.1186/s13007-019-0447-3DNA RNA extractionCTABSDSWoody speciesMacadamiaAvocado |
| spellingShingle | François F. Barbier Tinashe G. Chabikwa Muhammad U. Ahsan Stacey E. Cook Rosanna Powell Milos Tanurdzic Christine A. Beveridge A phenol/chloroform-free method to extract nucleic acids from recalcitrant, woody tropical species for gene expression and sequencing Plant Methods DNA RNA extraction CTAB SDS Woody species Macadamia Avocado |
| title | A phenol/chloroform-free method to extract nucleic acids from recalcitrant, woody tropical species for gene expression and sequencing |
| title_full | A phenol/chloroform-free method to extract nucleic acids from recalcitrant, woody tropical species for gene expression and sequencing |
| title_fullStr | A phenol/chloroform-free method to extract nucleic acids from recalcitrant, woody tropical species for gene expression and sequencing |
| title_full_unstemmed | A phenol/chloroform-free method to extract nucleic acids from recalcitrant, woody tropical species for gene expression and sequencing |
| title_short | A phenol/chloroform-free method to extract nucleic acids from recalcitrant, woody tropical species for gene expression and sequencing |
| title_sort | phenol chloroform free method to extract nucleic acids from recalcitrant woody tropical species for gene expression and sequencing |
| topic | DNA RNA extraction CTAB SDS Woody species Macadamia Avocado |
| url | http://link.springer.com/article/10.1186/s13007-019-0447-3 |
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