Optimization of CTAB-based RNA extraction for in planta Fusarium oxysporum f. sp. cubense gene expression study

Optimization of CTAB-based RNA extraction for in planta Fusarium oxysporum f. sp. cubense gene expression study

A crucial prerequisite for an insightful gene expression study is the quality of the nucleic acid extracted. High-quality nucleic acids allow comparative downstream analyses for both organisms during a phytopathogen infection. However, RNA extraction of pathogen-infected host materials usually invol...

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Main Authors: Poon, Nee Kiew, Rofina Yasmin Othman, Katharina Mebus, Teo, Chee How
Format: Article
Language:English
Published: Penerbit Universiti Kebangsaan Malaysia 2019
Online Access:http://journalarticle.ukm.my/14372/
http://journalarticle.ukm.my/14372/
http://journalarticle.ukm.my/14372/1/07%20Nee%20Kiew%20Poon.pdf
id ukm-14372
recordtype eprints
spelling ukm-143722020-03-10T02:24:34Z http://journalarticle.ukm.my/14372/ Optimization of CTAB-based RNA extraction for in planta Fusarium oxysporum f. sp. cubense gene expression study Poon, Nee Kiew Rofina Yasmin Othman, Katharina Mebus, Teo, Chee How A crucial prerequisite for an insightful gene expression study is the quality of the nucleic acid extracted. High-quality nucleic acids allow comparative downstream analyses for both organisms during a phytopathogen infection. However, RNA extraction of pathogen-infected host materials usually involves extraction methods that are optimised individually for either the pathogen or the host. Different sets of buffers or specialised commercial kits are often required. In this study, a streamlined CTAB-based extraction protocol was optimised for both the pure culture of Fusarium oxysporum f. sp. cubense (Foc) and infected banana roots. Foc cultures were grown on PDA overlaid by a nylon membrane and total nucleic acids were successfully extracted from mycelia with a ratio of 100 mg mycelia powder mass to 2 mL of CTAB buffer. Using the optimised protocol, LiCl-precipitated RNAs showed higher values of A260/280 (2.064 ± 0.021) and A260/230 (1.937 ± 0.076) compared to ethanol precipitated RNAs. Similar observation was observed for inoculated banana roots where LiCl-precipitated RNAs showed higher values of A260/280 and A260/230 compared to ethanol precipitated RNAs. qRT-PCR analysis using a pair of Foc specific primers, FoTEF1α, confirmed that the LiCl-precipitated RNA was more suitable for downstream gene expression studies. This extraction protocol is applicable for Foc in planta gene expression study with a high potential to be extended to other filamentous fungal pathogens. Penerbit Universiti Kebangsaan Malaysia 2019-10 Article PeerReviewed application/pdf en http://journalarticle.ukm.my/14372/1/07%20Nee%20Kiew%20Poon.pdf Poon, Nee Kiew and Rofina Yasmin Othman, and Katharina Mebus, and Teo, Chee How (2019) Optimization of CTAB-based RNA extraction for in planta Fusarium oxysporum f. sp. cubense gene expression study. Sains Malaysiana, 48 (10). pp. 2125-2133. ISSN 0126-6039 http://www.ukm.my/jsm/malay_journals/jilid48bil10_2019/KandunganJilid48Bil10_2019.htm
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institution Universiti Kebangasaan Malaysia
building UKM Institutional Repository
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language English
description A crucial prerequisite for an insightful gene expression study is the quality of the nucleic acid extracted. High-quality nucleic acids allow comparative downstream analyses for both organisms during a phytopathogen infection. However, RNA extraction of pathogen-infected host materials usually involves extraction methods that are optimised individually for either the pathogen or the host. Different sets of buffers or specialised commercial kits are often required. In this study, a streamlined CTAB-based extraction protocol was optimised for both the pure culture of Fusarium oxysporum f. sp. cubense (Foc) and infected banana roots. Foc cultures were grown on PDA overlaid by a nylon membrane and total nucleic acids were successfully extracted from mycelia with a ratio of 100 mg mycelia powder mass to 2 mL of CTAB buffer. Using the optimised protocol, LiCl-precipitated RNAs showed higher values of A260/280 (2.064 ± 0.021) and A260/230 (1.937 ± 0.076) compared to ethanol precipitated RNAs. Similar observation was observed for inoculated banana roots where LiCl-precipitated RNAs showed higher values of A260/280 and A260/230 compared to ethanol precipitated RNAs. qRT-PCR analysis using a pair of Foc specific primers, FoTEF1α, confirmed that the LiCl-precipitated RNA was more suitable for downstream gene expression studies. This extraction protocol is applicable for Foc in planta gene expression study with a high potential to be extended to other filamentous fungal pathogens.
format Article
author Poon, Nee Kiew
Rofina Yasmin Othman,
Katharina Mebus,
Teo, Chee How
spellingShingle Poon, Nee Kiew
Rofina Yasmin Othman,
Katharina Mebus,
Teo, Chee How
Optimization of CTAB-based RNA extraction for in planta Fusarium oxysporum f. sp. cubense gene expression study
author_facet Poon, Nee Kiew
Rofina Yasmin Othman,
Katharina Mebus,
Teo, Chee How
author_sort Poon, Nee Kiew
title Optimization of CTAB-based RNA extraction for in planta Fusarium oxysporum f. sp. cubense gene expression study
title_short Optimization of CTAB-based RNA extraction for in planta Fusarium oxysporum f. sp. cubense gene expression study
title_full Optimization of CTAB-based RNA extraction for in planta Fusarium oxysporum f. sp. cubense gene expression study
title_fullStr Optimization of CTAB-based RNA extraction for in planta Fusarium oxysporum f. sp. cubense gene expression study
title_full_unstemmed Optimization of CTAB-based RNA extraction for in planta Fusarium oxysporum f. sp. cubense gene expression study
title_sort optimization of ctab-based rna extraction for in planta fusarium oxysporum f. sp. cubense gene expression study
publisher Penerbit Universiti Kebangsaan Malaysia
publishDate 2019
url http://journalarticle.ukm.my/14372/
http://journalarticle.ukm.my/14372/
http://journalarticle.ukm.my/14372/1/07%20Nee%20Kiew%20Poon.pdf
first_indexed 2023-09-18T20:06:56Z
last_indexed 2023-09-18T20:06:56Z
_version_ 1777407221225750528

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