Extraction of High Quality RNA from Cannabis sativa Bast Fibres: A Vademecum for Molecular Biologists

Guerriero, Gea; Mangeot-Peter, Lauralie; Hausman, Jean-François; Legay, Sylvain

doi:10.3390/fib4030023

Cited by 13 publications

(12 citation statements)

References 17 publications

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“…When plants were approximately 1 m high, the top 20 cm of stems of three plants were harvested. Leaves that were larger than 10 mm (including petioles) were removed, the stem segments were homogenized in liquid nitrogen, and RNA was extracted using a modified CTAB extraction combined with an RNeasy Kit (Qiagen, Valencia, CA, USA) [15]. A total of 10 µg of RNA was sent to the Beijing Genome Institute (BGI) (BGI Inc., Shenzhen, China) for library preparation and paired-end transcriptome sequencing, as previously described [16].…”

Section: Methodsmentioning

confidence: 99%

Transcriptome Assembly of the Bast Fiber Crop, Ramie, Boehmeria nivea (L.) Gaud. (Urticaceae)

Al-Ani

Deyholos

2018

Fibers

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Ramie (Boehmeria nivea) is a perennial crop valued for its strong bast fibers. Unlike other major bast fiber crops, ramie fiber processing does not include retting, but does require degumming, suggesting distinctive features in pectin and the development and composition of fibers. A comprehensive transcriptome assembly of ramie has not been made available, to date. We obtained the sequence of RNA transcripts (RNA Seq) from the apical region of developing ramie stems and combined these with reads from public databases for a total of 157,621,051 paired-end reads (30.3 billion base pairs Gbp) used as input for de novo assembly, resulting in 70,721 scaffolds (≥200 base pairs (bp); N 50 = 1798 bp). As evidence of the quality of the assembly, 36,535 scaffolds aligned to at least one Arabidopsis protein (BLASTP e-value ≤ 10 −10 ). The resource described here for B. nivea will facilitate an improved understanding of bast fibers, cell wall, and middle lamella development in this and other comparative species.

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Section: Methodsmentioning

confidence: 99%

Transcriptome Assembly of the Bast Fiber Crop, Ramie, Boehmeria nivea (L.) Gaud. (Urticaceae)

Al-Ani

Deyholos

2018

Fibers

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“…A segment of 2.5 cm was collected from the middle of each internode to avoid too much variation in gene expression, due to the varying developmental stages of the cell types. Fibres were separated from the parenchyma/collenchyma cortical tissues by gently pressing the collected stem peels in 80% ethanol with a pestle, as described in [ 28 ]. The fibres were then quickly blotted dry using autoclaved wipers (WypAll, Kimberley-Clark, Muller & Wegener, Luxembourg, Grand Duchy of Luxembourg) and immediately frozen in liquid nitrogen.…”

Section: Methodsmentioning

confidence: 99%

Identification of Reference Genes for RT-qPCR Data Normalization in Cannabis sativa Stem Tissues

Mangeot-Peter

Legay

Hausman

et al. 2016

IJMS

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Gene expression profiling via quantitative real-time PCR is a robust technique widely used in the life sciences to compare gene expression patterns in, e.g., different tissues, growth conditions, or after specific treatments. In the field of plant science, real-time PCR is the gold standard to study the dynamics of gene expression and is used to validate the results generated with high throughput techniques, e.g., RNA-Seq. An accurate relative quantification of gene expression relies on the identification of appropriate reference genes, that need to be determined for each experimental set-up used and plant tissue studied. Here, we identify suitable reference genes for expression profiling in stems of textile hemp (Cannabis sativa L.), whose tissues (isolated bast fibres and core) are characterized by remarkable differences in cell wall composition. We additionally validate the reference genes by analysing the expression of putative candidates involved in the non-oxidative phase of the pentose phosphate pathway and in the first step of the shikimate pathway. The goal is to describe the possible regulation pattern of some genes involved in the provision of the precursors needed for lignin biosynthesis in the different hemp stem tissues. The results here shown are useful to design future studies focused on gene expression analyses in hemp.

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“…Since the rst application of CTAB-based extraction method for plants [22], there have been variations depending on the plant species. Although currently published methods on DNA extraction of abaca has been based mostly on the CTAB method of DNA extraction [9][10][11][12], several studies on extraction of DNA from the monocot crop palm [18] and RNA from the ber crop Cannabis sativa [23] have shown the effectiveness of using lysis buffers containing SDS. The use of SDS as the main detergent in the lysis buffer of Protocol 4 was also able to extract high molecular weight DNA from abaca leaf samples (Fig.…”

Section: Dna Extraction Methods Comparisonmentioning

confidence: 99%

“…Protocols having CTAB as the main detergent for lysis require an incubation time for at least 15 min at 65 o C. This ensures proper lysis of cells and sequestration of proteins and polysaccharides from the DNA [24,25]. Aside from CTAB, SDS is also an effective detergent in lysing plant tissues [18,23,26]. For Protocol 5, Triton X-100 was also added aside from CTAB as lysing reagent which is also an effective detergent for lysing cells [25].…”

Section: Dna Extraction Methods Comparisonmentioning

confidence: 99%

Extraction of high molecular weight abaca DNA suitable for next-generation sequencing

Koh

Barbosa²,

Aquino

et al. 2020

Preprint

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Background The abaca (Musa textilis Née) is a fiber crop native to the Philippines with high economic value because of its fiber - the Manila hemp, known to be the strongest of all the natural fibers. DNA extraction in abaca is difficult due to its fibrous nature, high cellulose content and polyphenol compounds. Thus an optimized DNA extraction method is required for extracting high quality abaca DNA for next-generation sequencing applications. Results In this study, we have compared five different methods for the extraction of high molecular weight DNA from abaca leaves. The methods are the traditional CTAB method (Protocol 1), the CTAB with PVP method (Protocol 2), the CTAB with 0.3% β-mercaptoethanol method (Protocol 3), SDS-method (Protocol 4) and CTAB with Triton X-100 and PVP method (Protocol 5). Out of the five methods tested, traditional CTAB-method (Protocol 1), CTAB with 0.3% β-mercaptoethanol method (Protocol 3) and SDS-method (Protocol 4) have shown to be the most consistent in giving high molecular weight DNA with good yield and purity based on A260/A280 and A260/A230 absorption values. TissueLyserII was also utilized for homogenization for the three extraction protocols for applications in high-throughput DNA extraction. DNA from two abaca varieties were extracted using the CTAB with 0.3% β-mercaptoethanol method (Protocol 3) and were sent for NGS based on Illumina HiSeq platform having both passed the quality control for library preparation. Conclusion The CTAB with 0.3% β-mercaptoethanol method (Protocol 3) was found to be the simplest and most consistent method for extracting average yield DNA with high quality for NGS applications. The SDS-method (Protocol 4) was determined to have the shortest processing time and together with TissueLyserII is the most appropriate method for high-throughput extraction of abaca samples which will be useful for genotyping-by-sequencing (GBS) studies.

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Extraction of High Quality RNA from Cannabis sativa Bast Fibres: A Vademecum for Molecular Biologists

Cited by 13 publications

References 17 publications

Transcriptome Assembly of the Bast Fiber Crop, Ramie, Boehmeria nivea (L.) Gaud. (Urticaceae)

Transcriptome Assembly of the Bast Fiber Crop, Ramie, Boehmeria nivea (L.) Gaud. (Urticaceae)

Identification of Reference Genes for RT-qPCR Data Normalization in Cannabis sativa Stem Tissues

Extraction of high molecular weight abaca DNA suitable for next-generation sequencing

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