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Since gene expression regulation underpins all developmental stages and is responsive to environmental signals, RNA profile analyses are critical to understanding the regulatory networks that govern biological processes. Many studies show that isolating high-quality RNA is a crucial step in this process. Transcription factors are the primary regulators of gene expression.
Adaptive main features can be attributed to mutations influencing regulatory genes or cis-sequences, which describe natural variation among organisms. Furthermore, epigenetic marks such as DNA cytosine methylation may be passed down across generations and may play a role in determining gene expression patterns.
The requirement for high-quality RNA and DNA from the same tissue sample in order to compare accurate gene expression with DNA sequence or epigenetic changes prompted the researchers to establish a methodology to meet this need. In order to prevent technological bias, a procedure that allows nucleic acid separation from various plant tissues and limited amounts of starting material is also desirable.
There are many techniques and commercial kits accessible; although, most of them are designed for animal tissues and do not allow for the simultaneous extraction of both types of nucleic acids. The procedure used in studies is based on phenol-chloroform phase separation, followed by lithium chloride (LiCl) and isopropanol precipitation of RNA and DNA, respectively.
The method was found to apply to a variety of Arabidopsis thaliana (Brassicaceae) tissues, including leaf, inflorescence stem, flower, fruit, cotyledon, seedlings, base, and embryo, as well as Avicennia schaueriana (Acanthaceae) tissues, including leaf, stem, and roots.
It was also successful in extracting RNA from Paspalum notatum (Poaceae) leaves and flowers, Theobroma cacao (Malvaceae) leaves, and Sorghum bicolour leaves (Poaceae). As a result, we conclude that this protocol could be extended to a wide range of plant species and could help with a variety of RNA and DNA-based analyses while avoiding sampling bias.
Plant Material
Unique tissues from A. thaliana ecotype Columbia-0 cultivated at 22°C under a long day-photoperiod (16 h light/8 h dark) have been used for RNA and DNA extractions. Plants cultivated in soil 60 days after germination (DAG) provided rosette leaf, caulinleaf, node, internode, vine, and fruit tissues, whereas cotyledons and rosette discs were derived from plants 21 and 30 DAG, respectively.
Protocol for simultaneous RNA and DNA extraction
From plants grown in MS/2 medium supplemented with 0.7 per cent sucrose, described obtaining embryo tissue from macerated seeds (2013).
Tissues were obtained directly from naturally growing plants for RNA and DNA extractions from A. schaueriana and P. notatum and plants grown in a greenhouse for S. bicolour and T. cacao.
RNA extraction methods that use phenol: chloroform phase separation and LiCl precipitation have been updated. DNA was extracted from the sample’s usually Manycauline leaf supernatant. The reasoning for the method mentioned here is based on the fact that when essential phenol, pH 8.0, is used for RNA extraction, DNA is retained in the inorganic process, and differential RNA precipitation by LiCl salt, resulting in differential RNA precipitation. As a result of the current procedure, high-quality RNA and DNA could be isolated from the same initial sample of different plant tissues.
On an agarose gel, the purity of nucleic acids was determined by the ratio of absorbance at 260 and 280 nm (A260/A280), with values of 1.8 suggesting extremely pure samples.
Evaluation of the efficiency of the extraction procedure and integrity of RNA and DNA
The method described in this study was found to be very effective for extracting nucleic acids from various A. thaliana tissues; on average, 20 g of total RNA and 1.6 g of DNA were obtained per sample, with an A260/A280 ratio of approximately 2.0 and no signs of degradation.
The tissues that were tested were a leaf, inflorescence stem, fruit, seedling, flower, core, cotyledon, and embryo; the nucleic acid yield varied between the last four described tissues and the overall RNA average, with flower yielding 2-3 folds more and embryo, cotyledon, and root yielding 2-4 folds less.
Similar findings were obtained for A. schaueriana leaf, stem, and root samples, as well as RNA extractions from T. cacao, S. bicolour, and P. notatum, demonstrating the protocol’s efficacy across various tissues and species. However, RNA extractions from the roots of soil-grown plants can contain impurities, and the samples should be filtered further using commercial columns before cDNA synthesis (Oliveria et al., 2015).
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Bibliography
Oliveria, R. R., Vianna, A.J.C., Reátegui, A.C.E., & Vincentz, M.G.A. (2015). An efficient method for simultaneous extraction of high-quality RNA and DNA from various plant tissues. Genetics & Molecular Research, 4(14), 4. https://sci-hub.se/https://pubmed.ncbi.nlm.nih.gov/26782533/
