ABSTRACT
BACKGROUND: High-purity RNA serves as the basic requirement for downstream molecular analysis of plant species, especially the differential expression of genes to various biotic and abiotic stimuli. But, the extraction of high-quality RNA is usually difficult from plants rich in polysaccharides and polyphenols, and their presence usually interferes with the downstream applications. The aim of the study is to optimize the extraction of high-quality RNA from diverse plant species/tissues useful for downstream molecular applications. RESULTS: Extraction of RNA using commercially available RNA extraction kits and routine hexadecyltrimethylammonium bromide (CTAB) methods did not yield good quality DNA-free RNA from Prosopis cineraria, Conocarpus erectus, and Phoenix dactylifera. A reliable protocol for the extraction of high-quality RNA from mature leaves of these difficult-to-extract trees was optimized after screening nine different methods. The DNase I-, and proteinase K treatment-free modified method, consisting of extraction with CTAB method followed by TRIzol, yielded high-quality DNA-free RNA with an A260/A280 and A260/A230 ratios > 2.0. Extraction of RNA from Conocarpus, the most difficult one, was successful by avoiding the heat incubation of ground tissue in a buffer at 65 oC. Pre-warming of the buffer for 5-10 min was sufficient to extract good-quality RNA. RNA integrity number of the extracted RNA samples ranged between 7 and 9.1, and the gel electrophoresis displayed intact bands of 28S and 18S RNA. A cDNA library constructed from the RNA of P. cineraria was used for the downstream applications. Real-time qPCR analysis using the cDNA from P. cineraria RNA confirmed the quality. The extraction of good quality RNA from samples of the desert-growing P. cineraria (> 20-years-old) collected in alternate months of the year 2021 (January to December covering winter, spring, autumn, and the very dry and hot summer) proved the efficacy of the protocol. The protocol's broad applicability was further validated by extracting good-quality RNA from 36 difficult-to-extract plant species, including tissues such as roots, flowers, floral organs, fruits, and seeds. CONCLUSIONS: The modified DNase I and Proteinase K treatment-free protocol enables to extract DNA-free, high-quality, intact RNA from a total of 39 difficult-to-extract plant species belonging to 32 angiosperm families is useful to extract good-quality RNA from dicots and monocots irrespective of tissue types and growing seasons.
ABSTRACT
High yield of good quality plasmid DNA from gram -ve bacteria (Agrobacterium tumefaciens, A. rhizogenes, and Rhizobium sp.) and gram +ve bacterium (Bacillus thuringiensis) is difficult. The widely used plasmid extraction kits for Escherichia coli yield a low quantity of poor-quality plasmid DNA from these species. We have optimized an in-house modification of the QIAprep Spin Miniprep kit protocol of Qiagen, consisting of two extraction steps. In the first, the centrifugation after adding neutralization buffer is followed by ethanol (absolute) precipitation of plasmid DNA. In the second extraction step, the precipitated DNA is dissolved in Tris-EDTA (TE) buffer, followed by an addition of 0.5 volumes of 5 M sodium chloride and 0.1 volumes of 20% (w/v) sodium dodecyl sulfate. After incubation at 65 °C for 15 min, the plasmid DNA is extracted with an equal volume of chloroform:isoamyl alcohol (CIA). RNase (20 mg/mL) is added to the upper phase retrieved after centrifugation and is incubated at 37 °C for 15 min. The extraction of the plasmid DNA with an equal volume of CIA is followed by centrifugation and is precipitated from the retrieved upper phase by adding an equal volume of absolute ethanol. The pellet obtained after centrifugation is washed twice with 70% (v/v) ethanol, air dried, dissolved in TE buffer, and quantified. This easy-to-perform protocol is free from phenol extraction, density gradient steps, and DNA binding columns, and yields high-quality plasmid DNA. The protocol opens an easy scale up to yield a large amount of high-quality plasmid DNA, useful for high-throughput downstream applications. Key features The protocol is free from density gradient steps and use of phenol. The protocol is an extension of the QIAprep Spin Miniprep kit (Qiagen) and is applicable for plasmid DNA isolation from difficult-to-extract bacterial species. The protocol facilitates the direct transformation of the ligation product into Agrobacterium by skipping the step of E. coli transformation. The plasmids isolated are of sequencing grade and the method is useful for extracting plasmids for metagenomic studies. Graphical overview Overview of the plasmid isolation protocol (modified QIAprep Spin Miniprep kit) of the present study.
