Rapid and Reliable Method of High-Quality RNA Extraction from Diverse Plants
American Journal of Plant Sciences
The isolation of high quality RNA is a crucial technique in plant molecular biology. The quality of RNA determines the reliability of downstream process like real time PCR. In this paper, we reported a high quality RNA extraction protocol for a variety of plant species. Our protocol is time effective than traditional RNA extraction methods. The method takes only an hour to complete the procedure. Spectral measurement and electrophoresis were used to demonstrate RNA quality and quantity. The
... d quantity. The extracted RNA was further used for cDNA synthesis, expression analysis and copy number determination through Real Time PCR. The results indicate that RNA was of good quality and fit for real time PCR. This high throughput plant RNA extraction protocol can be used to isolate high quality RNA from diverse plants for real time PCR and other downstream applications. et al. 3130 phenolics like tannis and fibrous tissues such as lignin (wood), that are difficult to remove and lysis  . The phenolic substances (i.e. Polysachharides and polyphenols) bind irreversibly with proteins and nucleic acids, leading to oxidation and degradation that makes it unsuitable for downstream applications -  . RNA extraction becomes difficult due to the presence of these secondary metabolites, that's why RNA extraction protocols needs modification. Number of methods for RNA extraction in various plant species has been developed, most of these are plant specific, many of these are practised for particular model organisms. So the use of these protocols for other non model species is somewhat limited . Method for total RNA isolation from durian tissues has been devised by Ky et al. . Ghawana et al.  described a phenol-based method for RNA isolation in plant species i.e. rich in secondary metabolites such as rheum (Rheum austral D. Don) and arnebia (Arnebia euchroma (Royle) I. M. Johnst.). Vasanthaiah et al.  devised the efficient protocol for functional RNA isolation in different grape tissue. Gehrig et al.  proposed a polyethylene glycol (PEG) based method to extract RNA from tissues with high presence of polysaccharides and polyphenols (e.g., Aloe L., Ananas Mill., Clusia L., Euphorbia L.). Many protocols have been proposed for RNA extraction from specific type of tissue, such as the trizol-based methods proposed for RNA extraction from siliques and seeds of Arabidopsis (DC) Heynh.  or the seeds of Davidia involucrata Baill.  . Many methods focus on rapid RNA isolation, but these are specially proposed for model organisms (e.g., Arabidopsis leaves) and are limited to PCR-based downstream applications (e.g., Berendzen et al. ). More than 50 references have been published for plant RNA extraction methods from last 3 years. Approximately 90% have been tested in only single plant lineage   and mostly are modification of the cetyltrimethylammonium bromide (CTAB) method with polyvinylpyrrolidone  . Recently, Yockteng et al.  published a high quality RNA extraction method for high quality RNA for diverse types of plants for gene expression analysis and next generation sequencing. Because of extraction techniques variability and the quality of their products, the comparative analysis of genomes or transcriptomes across plant lineages and tissue types remains a challenge for plant evolution researchers. Most isolation methods are complex and have low throughputs due to presence of polysaccharides and polyphenol. Thus, these methods produce low RNA yields. Real time PCR has been used as a major analytical platform for quantification of nucleic acid, due to its greater sensitivity and accuracy and better quantitative performance as compared to conventional PCR  . For real time PCR, it is important to isolate high quality RNA because impurities in RNA affect PCR amplification and influence the reliability of real time  . Although real time PCR provides increased high throughput and speed but high quality RNA isolation has lagged. Therefore a cost effective and high throughput method is needed to produce high quality RNA. Keeping in view the above facts, we have standardised a high throughput protocol which can be utilized for wide range of plant tissues across a broad range of taxa. We tested this protocol with different explants of different crops like cereals crops (rice, wheat, maize), vegetable crops (onion), commercial crop (sugarcane). Our protocol is safer, easiest and cost-efficient than other RNA extraction methods. The purpose of this study is to evaluate the efficiency of Trizol to isolate high yield and quality of RNA from different types of tissues. Our motive is to introduce this protocol and not to discount other protocols that work well in particular cases, but the aim is to present a single protocol that works well across a broad variety of plant tissue types and plant species within an hour. Materials and Methods Plant Materials Fresh leaves and callus of rice (Oryza sativa), leaf samples of wheat (Triticum aestivum), Wheat kernels (Triticum aestivum), roots of maize (Zea mays), leaf samples of onion (Allium cepa) and spindles of sugarcane (Saccharum officinalis) were collected from glasshouse of "School of Agricultural Biotechnology, Punjab Agricultural University, Ludhiana" and used for RNA extraction with following method.