DNA-free high-quality RNA extraction from 39 difficult-to-extract plant species (representing seasonal tissues and tissue types) of 32 families, and its validation for downstream molecular applications

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.

An Optimized and Cost-Effective RNA Extraction Method for Secondary Metabolite-Enriched Tissues of Norway Spruce (Picea abies)

“…The TRIzol reagent is a strong phenol-containing lysis solution that helps to disrupt cells while maintaining RNA integrity. However, in our study, the TRIzol-based extraction methods, including the standard TRIzol and the TRIzol-column hybrid approach, proved ineffective for isolating RNA from any tissue type of Norway spruce [15,33]. AGE showed the absence of visible bands in the RNA region, marked by substantial smearing across all lanes.…”

“…Successful RNA isolation is a crucial step in fundamental and applied research in molecular biology. Isolating high-quality RNA is essential for accurate downstream analyses, including gene expression studies, microarray analysis, RT-PCR, and RNA-Seq [15,26,27]. Obtaining a high-quality and quantity of RNA can be a significant challenge, and it often requires careful consideration of several factors [28].…”

“…In contrast, the original protocol suggests keeping the supernatant with ethanol for between 30 min and 2 h at −20 • C. These adjustments collectively enhance the efficiency of the RNA isolation process while maintaining reliable results. The major problem in dealing with secondary metaboliteenriched tissues is the oxidation of phenolic compounds, which form quinone/protein complexes and often co-precipitate with RNA [13,15]. This results in poor quality and a low yield.…”

“…In coniferous species, the low number of living cells in tissues and their highly lignified nature make cell disruption difficult [13]. Furthermore, high concentrations of polysaccharides, phenolics, and RNase-associated contaminations make isolating good-quality RNA difficult [8,14,15]. The phenolic compounds in tissues undergo oxidation to form quinones, which can irreversibly bind to nucleic acids and co-precipitate with RNA, ultimately resulting in RNA degradation and a low yield [5,8].…”

“…Different plant RNA extraction protocols employ guanidinium-thiocyanate-based, cetyltrimethylammonium bromide (CTAB)-based, or sodium dodecyl sulfate (SDS)-based approaches. However, it is crucial to optimize these methods for individual species and even for the characteristics of the target tissue [15,16]. Commercially available preparations use guanidium isothiocyanate and phenols to provide a high yield, but the quality remains low and is useful only for certain model plant systems [17].…”