Sains Malaysiana 36(1): 91-95 (2007)

Rapid Isolation of Total RNA from Conidia, Germinating Conidia

and Appressoria of the Fungal Plant Pathogen

Colletotrichum gloeosporioides

(Pengekstrakan pantas RNA jumlah daripada konidium, tiub percambahan

dan aperosium kulat patogen tumbuhan Colletotrichum gloeosporioides)

Nurhaida Kamaruddin, Farah Diba Abu Bakar, Rohaiza Ahmad Redzuan,

Nor Muhammad Mahadi & Abdul Munir Abdul Murad

School of Biosciences and Biotechnology

Faculty of Science and Technology, Universiti Kebangsaan Malaysia

43600 Bangi, Selangor, Malaysia

ABSTRACT

Effective and rapid methods for RNA extraction from conidia, germinating conidia and appressoria of the fungal plant pathogen, Colletotrichum gloeosporioides is reported in this study. The procedure for the RNA extraction from conidia and germinating conidia was carried out using TRI REAGENT^® solution (Molecular Research Center, USA) and can be completed in less than one and a half hours. The procedure for RNA extraction from appressoria was carried out using a modified protocol employing guanidine isothiocyanate and mechanical cell disruption by glass beads. The efficiency of the RNA extraction procedures was evaluated by several measures to determine RNA integrity, purity and applicability in RT-PCR. RNA integrity was assessed by observing the integrity of the major RNA species (18S and 28S rRNA) on denaturing agarose gel electrophoresis. The ethidium bromide-staining pattern of intact total RNA extracted from the three fungal morphogenetic cells showed clearly defined 28S and 18S rRNA bands and no genomic DNA contamination. Spectrophotometric assessment of RNA from each sample indicated relatively high purity and absence of carbohydrate contamination. Finally, we have demonstrated that the methods used for RNA extraction of conidia, germinating conidia and appressoria produced RNA of sufficient quality suitable for RT-PCR in detecting the expression of protein kinase A regulatory subunit gene in C. gloeosporioides.

Keywords: Colletotrichum gloeosporioides; RNA isolation; conidia; appressoria; germinating conidia

ABSTRAK

Dalam kajian ini kaedah pengekstrakan RNA yang pantas dan berkesan bagi konidium, tiub percambahan dan apresorium kulat patogen tumbuhan Colletotrichum gloeosporioides dilaporkan.Kaedah pengekstrakan RNA dari konidium dan tiub percambahan telah dijalankan menggunakan larutan TRI REAGENT^® (Molecular Research Center, USA) dan dapat diselesaikan dalam masa kurang daripada satu jam setengah. Pengekstrakan RNA dari apresorium telah dijalankan berdasarkan protokol yang telah diubahsuai dengan menggunakan guanidina isotiosianat dan manik kaca untuk pemecahan sel secara mekanikal. Keberkesanan kaedah-kaedah pengekstrakan RNA ini telah dinilai dengan beberapa kaedah pengukuran untuk menentukan integriti, ketulenan dan kesesuaian RNA yang telah dipencilkan dalam tindakbalas RT-PCR. Integriti RNA dinilai dengan mencerap integriti spesies utama RNA (18S dan 28S rRNA) pada elektroforesis gel agarosa ternyahasli. Corak jalur RNA jumlah yang diperoleh daripada tiga sel morfologi yang diwarnakan dengan etidium bromida menunjukkan jalur rRNA 28S dan 18S yang jelas serta bebas daripada kontaminasi DNA genom. Penilaian spektrofotometri RNA daripada setiap sampel menunjukkan ketulenan yang baik dan didapati bebas daripada kontaminasi karbohidrat. Akhirnya, kami menunjukkan bahawa kaedah yang digunakan untuk pengekstrakan konidium, tiub percambahan dan apresorium ini telah menghasilkan RNA yang berkualiti dan sesuai untuk digunakan dalam tindakbalas RT-PCR bagi mengesan pengekspresan gen subunit regulatori PKA C. gloeosporioides.

Kata kunci: Colletotrichum gloeosporioides; pengekstrakan RNA; konidium, apresorium; tiub percambahan

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