Improved CTAB method for RNA extraction of thick waxy leaf tissues from sago palm (Metroxylon sagu Rottb.)

Yan, Wei-Jie; Pendi, Fifi Hafizzah; Hussain, Hasnain

doi:10.1186/s40538-022-00329-9

Cited by 4 publications

(3 citation statements)

References 21 publications

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“…Total RNA was extracted from T. sebifera leaves using the CTAB method (Yan et al 2022). Having constructed and standardized cDNA libraries for the nine samples (three replicates for each of the three colour stages), these were sequenced using the Illumina Hiseq 6000 sequencing platform and 150‐bp paired‐end reads were generated.…”

Section: Methodsmentioning

confidence: 99%

Metabolome and Transcriptome Reveal Chlorophyll, Carotenoid, and Anthocyanin Jointly Regulate the Color Formation of Triadica sebifera

Liu,

Wang,

et al. 2024

Physiologia Plantarum

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The Chinese tallow tree (Triadica sebifera) is an economically important plant on account of its ornamental value and oil‐producing seeds. Leaf colour is a key characteristic of T. sebifera, with yellow‐, red‐ and purple‐leaved varieties providing visually impressive displays during autumn. In this study, we performed metabolomic and transcriptomic analyses to gain a better understanding of the mechanisms underlying leaf colour development in purple‐leaved T. sebifera at three stages during the autumnal colour transition, namely, green, hemi‐purple, and purple leaves. We accordingly detected 370 flavonoid metabolites and 10 anthocyanins, among the latter of which, cyanidin‐3‐xyloside and peonidin‐3‐O‐glucoside were identified as the predominant compounds in hemi‐purple and purple leaves. Transcriptomic analysis revealed that structural genes associated with the anthocyanin biosynthetic pathway, chlorophyll synthesis pathway and carotenoid synthesis pathway were significantly differential expressed at the three assessed colour stages. Additionally, transcription factors associated with the MYB‐bHLH‐WD40 complex, including 22 R2R3‐MYBs, 79 bHLHs and 44 WD40 genes, were identified as candidate regulators of the anthocyanin biosynthetic pathway. Moreover, on the basis of the identified differentially accumulated anthocyanins and key genes, we generated genetic and metabolic regulatory networks for anthocyanin biosynthesis in T. sebifera. These findings provide comprehensive information on the leaf transcriptome and three pigments of T. sebifera, thereby shedding new light on the mechanisms underlying the autumnal colouring of the leaves of this tree.

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Section: Methodsmentioning

confidence: 99%

Metabolome and Transcriptome Reveal Chlorophyll, Carotenoid, and Anthocyanin Jointly Regulate the Color Formation of Triadica sebifera

Liu,

Wang,

et al. 2024

Physiologia Plantarum

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“…Advances in next-generation sequencing technologies and bioinformatics have transformed our ability to explore the biology of plant growth and its reaction to environmental stimuli and stresses [5][6][7]. Understanding the fundamental processes of gene expression, signal transduction, gene regulation, and transcriptome analysis involves employing a diverse set of methodologies, including RNA sequencing (RNA-Seq), reverse transcription polymerase chain reaction (RT-PCR), and complementary DNA (cDNA) library preparation [8,9].…”

Section: Introductionmentioning

confidence: 99%

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

Singh,

Naseer,

Sellamuthu

et al. 2024

Plants

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Since the development of next-generation sequencing techniques and with the growing interest in transcriptomic studies, there is a demand for high-throughput RNA extraction techniques. General RNA extraction protocols are unreliable when it comes to the quality and quantity of isolated RNA obtained from different tissue types of different plant species. Despite Norway spruce (Picea abies) being one of the most significant and commercially valuable tree species in European forests, only limited genetic research is available. In this study, we developed a cetyltrimethylammonium bromide (CTAB) protocol by modifying the original method. We compared this CTAB protocol with other widely used methods for extracting RNA from different tissues (needle, phloem, and root) of Norway spruce, known for its richness in polyphenols, polysaccharides, and secondary metabolites. The modified CTAB method proves to be superior to the kit-based and TRIzol-based methods for extracting RNA from the metabolite-rich tissues of Norway spruce, resulting in high RNA quality and integrity values (RIN~7–9). The modified CTAB RNA extraction method is rapid, cost-effective, and relatively simple in yielding the desired RNA quality from Norway spruce tissues. It is optimal for RNA sequencing and other downstream molecular applications.

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“…The extraction of RNA, even from different species of the same genus, requires optimization for the protocol to yield high-quality, intact RNA. Various methods have been developed for RNA extraction from different plant tissues [5,6,11,12]. RNA extraction protocols across planta use guanidinium thiocyanate, CTAB, or sodium dodecyl sulfate (SDS) based methods [5,6,13,14].…”

Section: Introductionmentioning

confidence: 99%

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

et al. 2023

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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.

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Improved CTAB method for RNA extraction of thick waxy leaf tissues from sago palm (Metroxylon sagu Rottb.)

Cited by 4 publications

References 21 publications

Metabolome and Transcriptome Reveal Chlorophyll, Carotenoid, and Anthocyanin Jointly Regulate the Color Formation of Triadica sebifera

Metabolome and Transcriptome Reveal Chlorophyll, Carotenoid, and Anthocyanin Jointly Regulate the Color Formation of Triadica sebifera

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

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

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