Extraction of High-Quality, Melanin-Free RNA From Mycosphaerella fijiensis for cDNA Preparation

Abstract

High-quality RNA preparations are critical for further applications such as reverse transcriptase-polymerase chain reaction (RT-PCR) transcript amplifications, and elaboration of cDNA and expressed sequence tag libraries. Melanins are phenolic compounds present in many fungi and apparently play key roles in fungi pathogenesis and survival. However, during RNA extraction these compounds constitute a significant challenge to extraction of substantial quantities of high-quality RNA, and consequently to preparation of cDNA libraries. No method currently exists for RNA extraction from Mycosphaerella fijiensis that produces high quantities of melanin-free RNA. This fungus is the most important pathogen of cultivated Musa sp. varieties. A comparison is made between results obtained from the Trizol and RNeasy protocols for RNA extraction, two commercially available methods commonly used to obtain RNA from various sources. An improved methodology is described that allows isolation of intact RNA and elimination of melanins from M. fijiensis mycelium. RNA quality is evaluated by electrophoresis in formaldehyde-agarose gels, RT into cDNAs, and subsequent PCR amplification using primers designed against actin and beta- tubulin from fungi.

An Efficient Method for the Extraction of High-Quality Fungal Total RNA to Study the Mycosphaerella fijiensis–Musa spp. Interaction

“…RNA was also prepared using the RNeasy Ò Plant Mini Kit (QIAGEN, Germany) since this kit is recommended by the manufacturer for RNA purification from plants and filamentous fungi [8]. We also included the RNA extraction protocol for M. fijiensis reported by Islas-Flores et al [3]. In the first instance, M. fijiensis fresh samples were processed as described in the protocol [3].…”

“…We also included the RNA extraction protocol for M. fijiensis reported by Islas-Flores et al [3]. In the first instance, M. fijiensis fresh samples were processed as described in the protocol [3]. To allow a direct comparison of the RNA yield obtained, the Islas-Flores et al [3] protocol was also applied on lyophilized M. fijiensis mycelium.…”

“…Ethidium bromidestained agarose 1% gel electrophoresis (c): total RNA preparations from an infected plant [1] and fungal [2] material. (d): RT-PCR results using the M. fijiensis cytochrome b specific primer on total RNA preparations from a non-infected [1] and infected [2] 'Grande naine' leaf and fungal liquid culture [3]. Molecular weight marker: GeneRuler TM 100 bp plus DNA ladder, (Fermentas Life Sciences, Lithuania) [4] Plant Material…”

“…We also used the RNA extraction protocol for M. fijiensis reported by Islas-Flores et al [3] starting from 250 mg fresh mycelium. To allow direct comparison with our method, the protocol was also applied on 50 mg lyophilized mycelium (a weight ratio of fresh to dry fungal mycelium of 5 to 1 [8] to was taken into consideration).…”

“…Only one protocol for RNA isolation specific for M. fijiensis has been reported [3], which does not consider carbohydrate removal a crucial issue. Instead, this protocol focused on obtaining RNA preparations free of melanins, phenolic compounds naturally produced by many fungi.…”