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African Journal of Biotechnology

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A mini-scale hot borate method for the isolation of total RNA from a large number of cotton tissue samples

M Pang, JMcD Stewart, J Zhang

Abstract


Genetic genomics in cotton requires isolating RNA from a large number of plant tissue samples, in a mapping population. However, traditional methods for the extraction of RNA from cotton tissues is time consuming and not suitable for processing many samples at the same time. Here, we present a miniscale protocol for quick isolation of total RNA from cotton ovaries with high yield and quality suitable for gene expression studies. By modifying and scaling down a hot borate extraction method, a quick and easy method was developed to extract total RNA from ovaries of three genotypes ranging in ages from -1 to 10 days post anthesis (dpa) with an average yield of 600 μg/gfw (gram fresh weight) and ten dpa ovaries of 64 genotypes. The method was also successfully tested using cotton leaves and anthers. The quality and quantity of total RNA were suitable for reverse transcription-polymerase chain reaction (RT-PCR) and cDNA-amplified fragment length polymorphism (cDNA-AFLP) analysis.

Key words: Cotton, ovaries, RNA isolation, reverse transcription-polymerase chain reaction (RT-PCR), cDNAamplified fragment length polymorphism (cDNA-AFLP).




http://dx.doi.org/10.5897/AJB10.1425
AJOL African Journals Online