Optimization of CTAB-based RNA Extraction for in planta Fusarium oxysporum f. sp. cubense Gene Expression Study

Abstract

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 A 260/280 (2.064 ± 0.021) and A 260/230 (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 A 260/280 and A 260/230 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.Keywords: Fusarium oxysporum f. sp. cubense; hexadecyltrimethylammonium bromide (CTAB); in planta gene expression ABSTRAK Prasyarat penting untuk kajian pengekspresan gen yang tepat adalah kualiti asid nukleik yang diekstrak. Asid nukleik berkualiti tinggi membolehkan analisis hiliran komparatif bagi kedua-dua organisma semasa jangkitan fitopatogen. Walau bagaimanapun, pengambilan RNA perumah yang dijangkiti patogen biasanya melibatkan kaedah pengekstrakan yang dioptimumkan secara individu untuk sama ada patogen atau perumah. Pelbagai penimbal atau kit komersial khusus sering diperlukan. Dalam kajian ini, protokol pengekstrakan berasaskan CTAB dioptimumkan untuk kedua-dua kultur tulen Fusarium oxysporum f. sp. cubense (Foc) dan akar pisang yang dijangkiti. Foc dikultur pada PDA yang dilapisi oleh membran nilon dan asid nukleik berjaya diekstrak daripada miselium dengan nisbah 100 mg serbuk miselium kepada 2 mL penimbal CTAB. Dengan protokol yang dioptimumkan, RNA yang dimendak oleh LiCl menunjukkan nilai A 260/280 (2.064 ± 0.021) dan A 260/230 (1.937 ± 0.076) yang lebih tinggi berbanding RNA yang dimendak menggunakan etanol. Pemerhatian yang sama dicerap untuk akar pisang yang diinokulasi dengan RNA yang dimendak oleh LiCl menunjukkan nilai A 260/280 dan A 260/230 yang lebih tinggi berbanding RNA yang dimendak menggunakan etanol. Analisis qRT-PCR menggunakan pasangan pencetus khusus Foc, FoTEF1α, mengesahkan bahawa RNA yang dimendak oleh LiCl lebih sesuai untuk kajian pengekspresan gen hiliran. Protokol pengekstrakan ini boleh digunakan untuk k...

Optimization of CTAB-based RNA Extraction Method for Aspergillus flavus

“…Which form H-bond with polyphenol compound and this co-precipitate complex were removed or separated by high speed centrifugation by chloroform: isoamyl alcohol phase separation step (Poon et al, 2019).…”