Colloids and Surfaces B: Biointerfaces 54 (2007) 254–258 Short communication An efficient method for purifying high quality RNA from wheat pistils A. Manickavelu a,∗ , Kumiko Kambara b , Kohei Mishina a , Takato Koba a a Laboratory of Genetics and Plant Breeding, Faculty of Horticulture, Chiba University, 648, Matsudo, Chiba 271-8510, Japan b Laboratoire de Biologie Molecularie des Plantes Superieures (LAMPS), Department de Biologie Vegetale, 30 Quai Ernest-Ansermet, Universite de Geneve, CH-1211 Geneva 4, Switzerland Received 6 August 2006; received in revised form 20 October 2006; accepted 20 October 2006 Available online 27 October 2006 Abstract Many methods are available for total RNA extraction from plants, except the floral organs like wheat pistils containing high levels of polysaccha- rides that bind/or co-precipitate with RNA. In this protocol, a simple and effective method for extracting total RNA from small and feathery wheat pistils has been developed. Lithium chloride (LiCl) and phenol:chloroform:isoamylalcohol (PCI) were employed and the samples were ground in microcentrifuge tube using plastic pestle. A jacket of liquid nitrogen and simplified procedures were applied to ensure thorough grinding of the pistils and to minimize the samples loss. These measures substantially increased the recovery of total RNA (∼50%) in the extraction process. Reliable differential display by cDNA-AFLP was successfully achieved with the total RNA after DNase treatment and reverse transcription. This method is also practicable for gene expression and gene regulation studies in floral parts of other plants. © 2006 Elsevier B.V. All rights reserved. Keywords: cDNA-AFLP; Differential display; Reverse transcription; Total RNA; Wheat pistils 1. Introduction Molecular characterization by gene expression analysis in bread wheat (Triticum aestivum L.) genes is important because of its biological value, especially for crossability gene (Kr) in breeding point of view. It controls the interspecific hybridiza- tion with rye for further crop improvement. [1] reported that crossability is controlled by two loci, Kr1 and Kr2, where the dominant alleles reduces crossability, Kr1 being the more and Kr2 the less potent in effect. Extraction of high quality RNA is imperative for any biological studies viz., cDNA libraries, isolating genes by RT-PCR and investigating gene expression profiles by cDNA-AFLP [2]. The impediment for isolating sufficient quantities of high quality RNA from a floral tissue (e.g. wheat pistils) is yet to be solved particularly required for a tissue specific gene expression studies. A large number of plant RNA isolation procedures has been published [3,4], reflecting many difficulties especially for the successful isolation of both quantity and quality RNA. The main problems associated with plant nucleic acid isolation have been attributed primarily to the presence and co-precipitation of plant polysaccharides ∗ Corresponding author. Tel.: +81 8066372156. E-mail address: agromanicks@rediffmail.com (A. Manickavelu). and phenolic compounds [5–7]. These substances bind with RNA so as to render RNA unsuitable for cDNA synthesis, RT-PCR amplification, cDNA-AFLP and hybridization in northern blotting analyses. Thus, qualitative and quantitative differences in composition of polysaccharides and phenols in various plant tissues significantly alter the efficiency of nucleic acid extraction and purification procedures, which needs standardization and/or modification. The present study material, wheat pistils, is very small and feathery in nature. It contains a large amount of polysaccharides affecting RNA extraction and a specific method available for extracting high quality RNA from such a small bulk of materi- als (∼100 mg) is yet to develop. Efficient lysis of the cell and minimum sample losses are keys for such a method and it is important to take care of effective grinding in a pestle and mortar with liquid nitrogen. Hence, we designed an efficient method of extracting total RNA from any precious small amount of samples in 1.5 ml eppendorf tube for cDNA-AFLP transcriptional study. 2. Materials and methods 2.1. Plant material Based on the crossability percentage with rye (Dr. Koba, per- sonal communication), six low and four high crossability wheat 0927-7765/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.colsurfb.2006.10.024