Rapid isolation of total RNA from conidia, germinating conidia and appressoria of the fungal plant pathogen Colletotrichum gloeosporioides

Nurhaida Kamaruddin, Farah Diba Abu Bakar, Rohaiza Ahmad Redzuan, Nor Muhammad Mahadi, Abdul Munir Abd. Murad

Research output: Contribution to journalArticle

4 Citations (Scopus)

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.

Original languageEnglish
Pages (from-to)91-95
Number of pages5
JournalSains Malaysiana
Volume36
Issue number1
Publication statusPublished - Jul 2007

Fingerprint

appressoria
Glomerella cingulata
plant pathogens
conidia
RNA
purity
reverse transcriptase polymerase chain reaction
ribosomal RNA
guanidines
cAMP-dependent protein kinase
isothiocyanates
rapid methods
agarose
gel electrophoresis
glass
cells
carbohydrates
genomics

Keywords

  • Appressoria
  • Colletotrichum gloeosporioides
  • Conidia
  • Germinating conidia
  • RNA isolation

ASJC Scopus subject areas

  • General

Cite this

Rapid isolation of total RNA from conidia, germinating conidia and appressoria of the fungal plant pathogen Colletotrichum gloeosporioides. / Kamaruddin, Nurhaida; Abu Bakar, Farah Diba; Redzuan, Rohaiza Ahmad; Mahadi, Nor Muhammad; Abd. Murad, Abdul Munir.

In: Sains Malaysiana, Vol. 36, No. 1, 07.2007, p. 91-95.

Research output: Contribution to journalArticle

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