Name: non-aqueous isolation and enrichment of glandular capitate stalked and sessile trichomes from cannabis sativa
Uploaded: 2023-05-12T12:30:00
Description: Watch this Scientific Journal Video about Non-Aqueous Isolation and Enrichment of Glandular Capitate Stalked and Sessile Trichomes from Cannabis sativa at JoVE.com

The authors acknowledge financial support from CannabiVar Ltd. All plant material was generously provided by Professor Hinanit Koltai from the Volcani Center, Israel.

NOTE: The plant material used in this study consisted of four C. sativa ARO-Volcani strains (CS-11, CS-12, CS-13, and CS-14) that were grown in the Volcani Center, Israel, as described elsewhere11. Glandular capitate stalked trichomes were isolated from mature flowering inflorescences, and glandular capitate sessile trichomes were isolated from large fan leaves from mature non-flowering mother plants. All plant material was freshly picked and immediately stored at &#8722;80&#160;&#176;C.<...

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J., Mauleon, R., Mieog, J., Barkla, B. J., Kretzschmar, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+the+Cannabis+sativa+glandular+trichome+proteome.">Characterization of the Cannabis sativa glandular trichome proteome.</a> PLoS One. 16 (4), 0242633 (2021).</li><li>Liu, Y., Zhu, P., Cai, S., Haughn, G., Page, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three+novel+transcription+factors+involved+in+cannabinoid+biosynthesis+in+Cannabis+sativa+L.">Three novel transcription factors involved in cannabinoid biosynthesis in Cannabis sativa L.</a> Plant Molecular Biology. 106 (1-2), 49-65 (2021).</li><li>Slone, J. H., Kelsey, R. 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T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+functional+genomic+analysis+of+Solanum+glandular+trichome+types.">Comparative functional genomic analysis of Solanum glandular trichome types.</a> Plant Physiology. 155 (1), 524-539 (2011).</li><li>Bergau, N., Santos, A. N., Henning, A., Balcke, G. U., Tissier, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autofluorescence+as+a+signal+to+sort+developing+glandular+trichomes+by+flow+cytometry.">Autofluorescence as a signal to sort developing glandular trichomes by flow cytometry.</a> Frontiers in Plant Science. 7, 949 (2016).</li><li>Livingston, S. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cannabis+glandular+trichomes+alter+morphology+and+metabolite+content+during+flower+maturation.">Cannabis glandular trichomes alter morphology and metabolite content during flower maturation.</a> The Plant Journal. 101 (1), 37-56 (2019).</li><li>Turner, J. C., Hemphill, J. K., Mahlberg, P. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gland+distribution+and+cannabinoid+content+in+clones+of+Cannabis+sativa+L.">Gland distribution and cannabinoid content in clones of Cannabis sativa L.</a> American Journal of Botany. 64 (6), 687-693 (1977).</li><li>Turner, J. C., Hemphill, J. K., Mahlberg, P. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+determination+of+cannabinoids+in+individual+glandular+trichomes+of+Cannabis+sativa+L.+(Cannabaceae).">Quantitative determination of cannabinoids in individual glandular trichomes of Cannabis sativa L. (Cannabaceae).</a> American Journal of Botany. 65 (10), 1103-1106 (1978).</li><li>Hammond, C. T., Mahlberg, P. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphology+of+glandular+hairs+of+Cannabis+sativa+from+scanning+electron+microscopy.">Morphology of glandular hairs of Cannabis sativa from scanning electron microscopy.</a> American Journal of Botany. 60 (6), 524-528 (1973).</li><li>Marks, M. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+method+for+isolating+large+quantities+of+Arabidopsis+trichomes+for+transcriptome,+cell+wall,+and+other+types+of+analyses.">A new method for isolating large quantities of Arabidopsis trichomes for transcriptome, cell wall, and other types of analyses.</a> The Plant Journal. 56 (3), 483-492 (2008).</li><li>Huebbers, J. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+advanced+method+for+the+release,+enrichment+and+purification+of+high-quality+Arabidopsis+thaliana+rosette+leaf+trichomes+enables+profound+insights+into+the+trichome+proteome.">An advanced method for the release, enrichment and purification of high-quality Arabidopsis thaliana rosette leaf trichomes enables profound insights into the trichome proteome.</a> Plant Methods. 18 (1), 12 (2022).</li></ol>

Compared to the currently available trichome isolation protocols, two main modifications are described in the present protocol. These include the detachment of the trichomes from the plant material using dry ice in the initial step and substituting LN for the commonly used liquid buffer medium. The first modification for C. sativa trichome purification is based on an earlier protocol that introduced the use of crushed dry ice to detach the trichomes from geranium pedicels5. While traditio...

Glandular trichomes are hair-like structures present in plants that contain many secondary metabolites1 and represent a valuable bank of novel biosynthetic genes and enzymes2. In Cannabis, the biosynthesis of the important secondary metabolites, cannabinoids3 and terpenes4, is localized in the trichomes. Considering the role of trichomes in determining the quality of Cannabis both for medicinal and recreational uses, the study of trichome gene expression is of interest. To characterize the expression of trichome-specific genes, the trichomes of interest must first be isolated. Trichome isolation protocols were first described as early as 19925, and their latest developments have been recently reviewed2. In general, protocols for extracting glandular trichomes for transcriptomic characterization can be divided into two distinct sequential steps. The first step involves a thorough physical separation of the trichomes from the plant tissue. This step can be performed by using dry ice5, glass beads with a commercial apparatus6,7, grinding the plant material against a mesh sieve8, or vortexing the plant tissue in an isolation buffer9. The second step involves a more refined separation of the trichomes of interest from the microscopic plant residue and/or other trichome types. This step can be executed using density gradient centrifugation8,10 or sieves of various sizes7,9. Due to the extreme sensitivity of RNA in processed tissues to degrading agents, these two sequential steps are usually conducted in the ice-cold isolation medium, often in the presence of protein inhibitors4.
Conventional trichome isolation protocols require, in addition to the ice-cold temperatures, large amounts of isolation medium to ensure an efficient extraction procedure. The combination of these components results in an arduous, time-consuming isolation process that hinders high throughput. Presenting a straightforward, user-friendly alternative trichome isolation protocol is, therefore, likely to be beneficial for various aspects associated with trichome characterization. The present paper aims to offer an alternative protocol for isolating stalked and sessile glandular capitate trichomes from Cannabis sativa by combining and integrating several elements from the conventional protocols. These elements include dry ice5, the passing of the trichomes through several micro-sieves with decreasing pore sizes7,9, and substituting liquid nitrogen (LN)&#160;for the isolation medium8.
The novelty of the present trichome isolation protocol, as compared to conventional protocols, presents in a number of ways. This protocol is convenient, as it does not require hazardous components. The procedure can be conducted in the lab with minimal precautions and facilitates high throughput. Substituting LN for the standard liquid isolation medium ensures the integrity of the trichomes throughout the isolation process, enabling subsequent transcriptomic analysis. Upon sublimation of the LN and dry ice, the isolated trichomes are left free of harmful residuals. Further, the propensity of LN to sublimate at room temperature allows its generous use throughout the protocol. In contrast, using large volumes of conventional isolation medium generates practical difficulties in its handling. Finally, the protocol decreases the separation of the disc cell from the remaining fragile head structure of the glandular trichome, enabling the retention of the headspace content.
This protocol is presented in a detailed step-by-step fashion designed to assist the technical practice of isolating C. sativa glandular capitate trichomes. The protocol provides a manageable workflow that results in isolated trichomes with a high concentration and purity that are appropriate for downstream molecular analysis.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Bioanalyzer RNA Pico 6000 chip</td>
			<td>Agilent, Germany</td>
			<td>Reorder number 5067-1513</td>
			<td>Lab-on-a-chip system&#160;</td>
		</tr>
		<tr>
			<td>Transsonic-310</td>
			<td>Elma, Germany</td>
			<td>D-78224</td>
			<td>Ultrasonic cleaning unit&#160;</td>
		</tr>
		<tr>
			<td>TruSeq RNA Sample Prep Kit v2</td>
			<td>Illumina, USA</td>
			<td>RS-122-2001</td>
			<td>Sample preperation for RNA sequencing library</td>
		</tr>
		<tr>
			<td>Spectrum Plant Total RNA Kit&#160;</td>
			<td>SIGMA-ALDRICH, USA</td>
			<td>STRN50-1KT</td>
			<td>Plant Total RNA Kit&#160;</td>
		</tr>
		<tr>
			<td>Nylon micro-sieve with a mesh size of 350 &#181;m (40 x 40 cm or larger than the circumference of the flour sifter)</td>
			<td>Sinun Tech, Israel</td>
			<td>r0350n350210</td>
			<td>Nylon&#160;screen aperture</td>
		</tr>
		<tr>
			<td>Nylon micro-sieve with mesh size of 150 &#181;m (size of 30 x 30 cm)</td>
			<td>Sinun Tech, Israel</td>
			<td>r0150n360465</td>
			<td>Nylon&#160;screen aperture</td>
		</tr>
		<tr>
			<td>Nylon micro-sieve with mesh size o 105 &#181;m (size of 30 x 30 cm)</td>
			<td>Sinun Tech, Israel</td>
			<td>r0105n320718</td>
			<td>Nylon&#160;screen aperture</td>
		</tr>
		<tr>
			<td>Nylon micro-sieve with mesh size o 80 &#181;m (size of 30 x 30 cm)</td>
			<td>Sinun Tech, Israel</td>
			<td>r0080n370465</td>
			<td>Nylon&#160;screen aperture</td>
		</tr>
		<tr>
			<td>Nylon micro-sieve with mesh size o 65 &#181;m (size of 30 x 30 cm)</td>
			<td>Sinun Tech, Israel</td>
			<td>r0065n340715</td>
			<td>Nylon&#160;screen aperture</td>
		</tr>
		<tr>
			<td>Nylon micro-sieve with mesh size o 50 &#181;m (size of 30 x 30 cm)</td>
			<td>Sinun Tech, Israel</td>
			<td>r0080n370465</td>
			<td>Nylon&#160;screen aperture</td>
		</tr>
		<tr>
			<td>Up to 10 g of frozen plant material (stored in -80 oC or liquid nitrogen)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Suitable gloves for handling low temperatures</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Safety goggles</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>1 mm screen door (mosquito) mesh (strip of 30 x 100 cm)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Large strainer (colander) with holes approximately 5 mm</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>1 L glass beaker</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>1 block of dry ice (0.5-1 kg)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Hammer and hard flat object</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Two 5 L plastic containers</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Rubber bands</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Large flour sifter or sieve strainer- preferably one with a detachable plastic ring on the circumference</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Several large and small round bottom stainless steel containers. One of them should be larger than the flour sifter&#39;s circumference (approximately 40 cm in diameter), to minimize the loss of the sifted mass outside the round bottome stainless steel container</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Pre-chilled (via liquid nitrogen) stainless steel spoon, spatula, and scoopula</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Clean plate</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Several clothespins</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Pre-chilled (via liquid nitrogen) labeled 1.5 mL tubes with holes poked on the lid with a sterile needle</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Two containers of liquid nitrogen</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>1 cm wide painting brush</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

non-aqueous isolation and enrichment of glandular capitate stalked and sessile trichomes from cannabis sativa

This paper presents a protocol for the convenient and high-throughput isolation and enrichment of glandular capitate stalked and sessile trichomes from Cannabis sativa. The biosynthetic pathways for cannabinoid and volatile terpene metabolism are localized primarily in the Cannabis trichomes, and isolated trichomes are beneficial for transcriptome analysis. The existing protocols for isolating glandular trichomes for transcriptomic characterization are inconvenient and deliver compromised trichome heads and a relatively low amount of isolated trichomes. Furthermore, they rely on expensive apparatus and isolation media containing protein inhibitors to avoid RNA degradation. The present protocol suggests combining three individual modifications to obtain a large amount of isolated glandular capitate stalked and sessile trichomes from C. sativa mature female inflorescences and fan leaves, respectively. The first modification involves substituting liquid nitrogen for the conventional isolation medium to facilitate the passage of trichomes through the micro-sieves. The second modification involves using dry ice to detach the trichomes from the plant source. The third modification involves passing the plant material consecutively through five micro-sieves of diminishing pore sizes. Microscopic imaging demonstrated the effectiveness of the isolation technique for both trichome types. In addition, the quality of RNA extracted from the isolated trichomes was appropriate for downstream transcriptomic analysis.

The main modification included in this protocol compared to conventional trichome isolation protocols is substituting the standard isolation medium with LN. Using LN as an isolation medium allows a relaxed workflow, because as long as the samples are submerged in LN, metabolic degradation is not likely to occur. Furthermore, as the protocol avoids the hazardous components (i.e., aurintricarboxylic acid and &#946;-mercaptoethanol) used in traditional trichome isolation medium, the work is not restricted to a chemical hood...

Watch this Scientific Journal Video about Non-Aqueous Isolation and Enrichment of Glandular Capitate Stalked and Sessile Trichomes from Cannabis sativa at JoVE.com

Non-Aqueous Isolation and Enrichment of Glandular Capitate Stalked and Sessile Trichomes from Cannabis sativa

A protocol is presented for the convenient and high-throughput isolation and enrichment of glandular capitate stalked and sessile trichomes from Cannabis sativa. The protocol is based on a dry, non-buffer extraction of trichomes using only liquid nitrogen,&#160;dry ice, and nylon sieves and is suitable for RNA extraction and transcriptomic analysis.

Non-Aqueous Isolation and Enrichment of Glandular Capitate Stalked and Sessile Trichomes from Cannabis sativa

This paper presents a protocol for the convenient and high-throughput isolation and enrichment of glandular capitate stalked and sessile trichomes from ...

The significance of the protocol lies in its convenience in the short time needed for its completion. This enables a high throughput, a high concentration, and a high degree of the isolation of the stalked and sessile trichomes. The main advantage of this technique is its straightforwardness.During this technique, one should consider the trichome density of the plant source and inspect the isolation degree at each step via microscope while performing the protocol for the first time. Demonstrating this technique will be Nurit Shalev, an expert on cannabis from Volcani Center. Begin by crushing a dry ice block into small, fine flakes using a hammer and a hard, flat object in a five liter plastic container.Use a large strainer, and sieve the fine dry ice flakes from the non-crushed dry ice into another five liter plastic container. Pour approximately 200 cubic centimeters of the dry ice flakes into an upright one liter glass beaker. Add up to 10 grams of frozen cannabis sativa inflorescences onto the first layer of crushed dry ice, and cover it with an additional layer of 200 cubic centimeters of finely crushed dry ice.Then cover the opening of the one liter glass beaker with two to three layers of a one millimeter screen door mosquito net, and secure it with rubber bands. Next, pour liquid nitrogen into a large round-bottom stainless steel container. Insert a 350 micrometer mesh in a flour sifter or sieve strainer to cover the flour sifter mesh from below.Place this flour sifter above the large round-bottom stainless steel container filled with liquid nitrogen. To minimize the loss of the sifted mass, ensure that the container's width exceeds that of the flour sifter. Separate the trichomes from the plant material by shaking the one liter glass with its opening pointing downward toward the flour sifter.Set aside the beaker every two to three minutes, allowing the sifting of the crushed dry ice and plant material accumulated on the flour sieve. Sift the flour sieve horizontally to facilitate the passage of the plant material into the liquid nitrogen in the round-bottom stainless steel container placed below. Next, add liquid nitrogen to the stainless steel container and the crushed dry ice to the one liter glass beaker when their levels run low.When the plant material on the flour sieve is depleted, refill the used plant material in the glass beaker with an additional 10 grams of fresh plant material, and repeat the sifting process until the collection of a sufficient amount of enriched trichomes in the container. Verify the present of a white powder-like substance at the bottom of the stainless steel container submerged in liquid nitrogen before a microscope observation to confirm the collection of sufficient trichomes. Add a small amount of liquid nitrogen to a clean, small round-bottom stainless steel container.Then fold a 40 by 40 centimeter microsieve having a 150 micrometer mesh size twice to obtain a 20 by 20 centimeter fold, and open it in a cone-like shape. Secure the mesh cone to the edge of the round-bottom stainless steel container with one or two clothespins so that the opened part of the cone is upright, and it's pointed part is partly submerged in liquid nitrogen. Once done, gently pour the liquid nitrogen containing the white powder-like substance obtained earlier into the microsieve cone.Using a wide brush, gather and transfer any remaining plant material from the first container into the 150 micrometer mesh cone. Next, carefully detach the clothespin from the lid of the container, and open the microsieve cone to locate the trichomes in the middle of the opened microsieve. Hold all four corners of the microsieve together, and submerge the middle part containing the trichomes in liquid nitrogen.Gently dip and shake the microsieve in the liquid nitrogen for one minute. Using a sterile needle, make tiny holes in the cap of a 50 milliliter test tube to avoid pressure building. Scoop the non-sifted plant debris, dry ice, and larger trichomes wrapped in the center of the microsieve into a 50 milliliter test tube, and store it at minus 80 degrees Celsius for future use.Transfer the sifted trichomes submerged in the liquid nitrogen sequentially through microsieves, having decreasing pour sizes from 105 to 80 to 65, and then to 50 micrometer. After each transfer, collect the different sized trichomes remaining on the microsieve separately, and label them. Make tiny holes in the cap of the tube using a sterile needle.Transfer the desired trichomes submerged in liquid nitrogen and wrapped in the 50 micrometer microsieve to a clean plate using a pre-chilled spoon. Then quickly transfer the powder-like trichomes into a labeled pre-chilled 1.5 milliliter tube using a pre-chilled spatula, and immediately store the tubes at minus 80 degrees Celsius for further studies. In the initial stage of the isolation process, pieces of leaf tissue were predominant in the isolated portion, in contrast to the final isolation product, which was composed entirely of non-contaminated isolated trichomes.The quality of the isolated granular capitate stalked and sessile trichomes was observed in the final isolation step. The purity of the trichomes isolated using this method was comparable with a conventional protocol. The delicate stalked trichome head structure was retained, and the whole heads of the isolated stalked trichome were visible, in contrast to the traditional method, where the complete trichome head structure was missing, and only disc cells were present.The RNA extraction and estimation of RNA integrity of the isolated trichomes indicated high RNA integrity number or RIN values from 9.4 to 10, and gel chromatography indicated high RNA integrity. Carefully perform the T bag-like infusion motion of the 150 to 50 micro microsieved isolation. This method can provide insights into the transcriptomic and probably proteomic makeup of trichomes from cannabis and trichomes of other plant species.Following this procedure, metabolic and transcriptomic characterizations can be performed. These additional methods can help to understand the expression levels of the genes in the isolated trichomes and their metabolic profiles. This technique has enabled the transcriptomic characterization of glandular trichomes from different cannabis varieties.

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Genetics

Isolation of Giant Lampbrush Chromosomes from Living Oocytes of Frogs and Salamanders

We describe methods for studying the giant transcriptionally active lampbrush chromosomes (LBCs) found in the oocyte, or unlaid egg, of frogs and salamanders. Individual LBCs can be up to 1 mm in length and they reside in a gigantic nucleus, itself up to 0.5 mm in diameter. The large size of the chromosomes permits unparalleled observations of active genes by light optical microscopy, but at the same time special techniques are required for isolating the nucleus, removing the nuclear envelope, and spreading the chromosomes on a microscope slide. The oocyte nucleus, also called the germinal vesicle (GV), is isolated in a medium that allows partial gelling of the nuclear actin and preserves the delicate structure of the LBCs. This step is carried out manually under a dissecting microscope using jeweler&#39;s forceps. Next, the nuclear envelope is removed, again manually with jeweler&#39;s forceps. The nuclear contents are quickly transferred to a medium that disperses the actin gel and allows the undamaged LBCs to settle onto a microscope slide. At this point the LBCs and other nuclear organelles can be viewed by phase contrast or differential interference contrast microscopy, although finer details are obscured by Brownian motion. For high resolution microscopical observation or molecular analysis, the whole preparation is centrifuged to attach the delicate LBCs firmly to the slide. A brief fixation in paraformaldehyde is then followed by immunofluorescent staining or in situ hybridization. LBCs are in a transcriptionally active state and their enormous size permits molecular analysis at the individual gene level using confocal or super-resolution microscopy.

We present simple techniques for isolating giant transcriptionally active lampbrush chromosomes from living oocytes of frogs and salamanders. We describe how to observe these chromosomes &#34;alive&#34; by phase contrast or differential interference contrast, and how to fix them for fluorescent in situ hybridization or immunofluorescent staining.

We describe methods for studying the giant transcriptionally active lampbrush chromosomes (LBCs) found in the oocyte, or unlaid egg, of frogs and ...

A Robotic Platform for High-throughput Protoplast Isolation and Transformation

Over the last decade there has been a resurgence in the use of plant protoplasts that range from model species to crop species, for analysis of signal transduction pathways, transcriptional regulatory networks, gene expression, genome-editing, and gene-silencing. Furthermore, significant progress has been made in the regeneration of plants from protoplasts, which has generated even more interest in the use of these systems for plant genomics. In this work, a protocol has been developed for automation of protoplast isolation and transformation from a &#39;Bright Yellow&#39; 2 (BY-2) tobacco suspension culture using a robotic platform. The transformation procedures were validated using an orange fluorescent protein (OFP) reporter gene (pporRFP) under the control of the Cauliflower mosaic virus 35S promoter (35S). OFP expression in protoplasts was confirmed by epifluorescence microscopy. Analyses also included protoplast production efficiency methods using propidium iodide. Finally, low-cost food-grade enzymes were used for the protoplast isolation procedure, circumventing the need for lab-grade enzymes that are cost-prohibitive in high-throughput automated protoplast isolation and analysis. Based on the protocol developed in this work, the complete procedure from protoplast isolation to transformation can be conducted in under 4 hr, without any input from the operator. While the protocol developed in this work was validated with the BY-2 cell culture, the procedures and methods should be translatable to any plant suspension culture/protoplast system, which should enable acceleration of crop genomics research.

A high-throughput, automated, tobacco protoplast production and transformation methodology is described. The robotic system enables massively parallel gene expression and discovery in the model BY-2 system that should be translatable to non-model crops.

Over the last decade there has been a resurgence in the use of plant protoplasts that range from model species to crop species, for analysis of signal ...

From a Natural Product to Its Biosynthetic Gene Cluster: A Demonstration Using Polyketomycin from Streptomyces diastatochromogenes T&#252;6028

Streptomyces strains are known for their capability to produce a lot of different compounds with various bioactivities. Cultivation under different conditions often leads to the production of new compounds. Therefore, production cultures of the strains are extracted with ethyl acetate and the crude extracts are analyzed by HPLC. Furthermore, the extracts are tested for their bioactivity by different assays. For structure elucidation the compound of interest is purified by a combination of different chromatography methods.
Genome sequencing coupled with genome mining allows the identification of a natural product biosynthetic gene cluster using different computer programs. To confirm that the correct gene cluster has been identified, gene inactivation experiments have to be performed. The resulting mutants are analyzed for the production of the particular natural product. Once the correct gene cluster has been inactivated, the strain should fail to produce the compound.
The workflow is shown for the antibacterial compound polyketomycin produced by Streptomyces diastatochromogenes T&#252;6028. Around ten years ago, when genome sequencing was still very expensive, the cloning and identification of a gene cluster was a very time-consuming process. Fast genome sequencing combined with genome mining accelerates the trial of cluster identification and opens up new ways to explore biosynthesis and to generate novel natural products by genetic methods. The protocol described in this paper can be assigned to any other compound derived from a Streptomyces strain or another microorganism.

From a Natural Product to Its Biosynthetic Gene Cluster: A Demonstration Using Polyketomycin from &lt;em&gt;Streptomyces diastatochromogenes&lt;/em&gt; T&amp;#252;6028

Here we present a detailed protocol of (A) the identification of a natural product with antibiotic activity, (B) the purification of the compound, (C) the first model of its biosynthesis, (D) genome sequencing/-mining and the (E) verification of the biosynthetic gene cluster.

Streptomyces strains are known for their capability to produce a lot of different compounds with various bioactivities. Cultivation under different ...

Optimization and Comparative Analysis of Plant Organellar DNA Enrichment Methods Suitable for Next-generation Sequencing

Plant organellar genomes contain large, repetitive elements that may undergo pairing or recombination to form complex structures and/or sub-genomic fragments. Organellar genomes also exist in admixtures within a given cell or tissue type (heteroplasmy), and an abundance of subtypes may change throughout development or when under stress (sub-stoichiometric shifting). Next-generation sequencing (NGS) technologies are required to obtain deeper understanding of organellar genome structure and function. Traditional sequencing studies use several methods to obtain organellar DNA: (1) If a large amount of starting tissue is used, it is homogenized and subjected to differential centrifugation and/or gradient purification. (2) If a smaller amount of tissue is used (i.e., if seeds, material, or space is limited), the same process is performed as in (1), followed by whole-genome amplification to obtain sufficient DNA. (3) Bioinformatics analysis can be used to sequence the total genomic DNA and to parse out organellar reads. All these methods have inherent challenges and tradeoffs. In (1), it may be difficult to obtain such a large amount of starting tissue; in (2), whole-genome amplification could introduce a sequencing bias; and in (3), homology between nuclear and organellar genomes could interfere with assembly and analysis. In plants with large nuclear genomes, it is advantageous to enrich for organellar DNA to reduce sequencing costs and sequence complexity for bioinformatics analyses. Here, we compare a traditional differential centrifugation method with a fourth method, an adapted CpG-methyl pulldown approach, to separate the total genomic DNA into nuclear and organellar fractions. Both methods yield sufficient DNA for NGS, DNA that is highly enriched for organellar sequences, albeit at different ratios in mitochondria and chloroplasts. We present the optimization of these methods for wheat leaf tissue and discuss major advantages and disadvantages of each approach in the context of sample input, protocol ease, and downstream application.

The comparison and optimization of two plant organellar DNA enrichment methods are presented: traditional differential centrifugation and fractionation of the total gDNA based on methylation status. We assess the resulting DNA quantity and quality, demonstrate performance in short-read next-generation sequencing, and discuss the potential for use in long-read single-molecule sequencing.

Plant organellar genomes contain large, repetitive elements that may undergo pairing or recombination to form complex structures and/or sub-genomic ...

Sample Preparation and Analysis of RNASeq-based Gene Expression Data from Zebrafish

The analysis of global gene expression changes is a valuable tool for identifying novel pathways underlying observed phenotypes. The zebrafish is an excellent model for rapid assessment of whole transcriptome from whole animal or individual cell populations due to the ease of isolation of RNA from large numbers of animals. Here a protocol for global gene expression analysis in zebrafish embryos using RNA sequencing (RNASeq) is presented. We describe preparation of RNA from whole embryos or from cell populations obtained using cell sorting in transgenic animals. We also describe an approach for analysis of RNASeq data to identify enriched pathways and Gene Ontology (GO) terms in global gene expression data sets. Finally, we provide a protocol for validation of gene expression changes using quantitative reverse transcriptase PCR (qRT-PCR). These protocols can be used for comparative analysis of control and experimental sets of zebrafish to identify novel gene expression changes, and provide molecular insight into phenotypes of interest.

This protocol presents an approach for whole transcriptome analysis from zebrafish embryos, larvae, or sorted cells. We include isolation of RNA, pathway analysis of RNASeq data, and qRT-PCR-based validation of gene expression changes.

The analysis of global gene expression changes is a valuable tool for identifying novel pathways underlying observed phenotypes. The zebrafish is an ...

Robust DNA Isolation and High-throughput Sequencing Library Construction for Herbarium Specimens

Herbaria are an invaluable source of plant material that can be used in a variety of biological studies. The use of herbarium specimens is associated with a number of challenges including sample preservation quality, degraded DNA, and destructive sampling of rare specimens. In order to more effectively use herbarium material in large sequencing projects, a dependable and scalable method of DNA isolation and library preparation is needed. This paper demonstrates a robust, beginning-to-end protocol for DNA isolation and high-throughput library construction from herbarium specimens that does not require modification for individual samples. This protocol is tailored for low quality dried plant material and takes advantage of existing methods by optimizing tissue grinding, modifying library size selection, and introducing an optional reamplification step for low yield libraries. Reamplification of low yield DNA libraries can rescue samples derived from irreplaceable and potentially valuable herbarium specimens, negating the need for additional destructive sampling and without introducing discernible sequencing bias for common phylogenetic applications. The protocol has been tested on hundreds of grass species, but is expected to be adaptable for use in other plant lineages after verification. This protocol can be limited by extremely degraded DNA, where fragments do not exist in the desired size range, and by secondary metabolites present in some plant material that inhibit clean DNA isolation. Overall, this protocol introduces a fast and comprehensive method that allows for DNA isolation and library preparation of 24 samples in less than 13 h, with only 8 h of active hands-on time with minimal modifications.

This article demonstrates a detailed protocol for DNA isolation and high-throughput sequencing library construction from herbarium material including rescue of exceptionally poor-quality DNA.

Herbaria are an invaluable source of plant material that can be used in a variety of biological studies. The use of herbarium specimens is associated with ...

Isolation, Characterization and MicroRNA-based Genetic Modification of Human Dental Follicle Stem Cells

To date, several stem cell types at different developmental stages are in the focus for the treatment of degenerative diseases. Yet, certain aspects, such as initial massive cell death and low therapeutic effects, impaired their broad clinical translation. Genetic engineering of stem cells prior to transplantation emerged as a promising method to optimize therapeutic stem cell effects. However, safe and efficient gene delivery systems are still lacking. Therefore, the development of suitable methods may provide an approach to resolve current challenges in stem cell-based therapies.
The present protocol describes the extraction and characterization of human dental follicle stem cells (hDFSCs) as well as their non-viral genetic modification. The postnatal dental follicle unveiled as a promising and easily accessible source for harvesting adult multipotent stem cells possessing high proliferation potential. The described isolation procedure presents a simple and reliable method to harvest hDFSCs from impacted wisdom teeth. Also this protocol comprises methods to define stem cell characteristics of isolated cells. For genetic engineering of hDFSCs, an optimized cationic lipid-based transfection strategy is presented enabling highly efficient microRNA introduction without causing cytotoxic effects. MicroRNAs are suitable candidates for transient cell manipulation, as these small translational regulators control the fate and behavior of stem cells without the hazard of stable genome integration. Thus, this protocol represents a safe and efficient procedure for engineering of hDFSCs that may become important for optimizing their therapeutic efficacy.

This protocol describes the transient genetic engineering of dental stem cells extracted from the human dental follicle. The applied non-viral modification strategy may become a basis for the improvement of therapeutic stem cell products.

To date, several stem cell types at different developmental stages are in the focus for the treatment of degenerative diseases. Yet, certain aspects, such ...

Isolating, Sequencing and Analyzing Extracellular MicroRNAs from Human Mesenchymal Stem Cells

Extracellular and circulating RNAs (exRNA) are produced by many cell types of the body and exist in numerous bodily fluids such as saliva, plasma, serum, milk and urine. One subset of these RNAs are the posttranscriptional regulators &#8211; microRNAs (miRNAs). To delineate the miRNAs produced by specific cell types, in vitro culture systems can be used to harvest and profile exRNAs derived from one subset of cells. The secreted factors of mesenchymal stem cells are implicated in alleviating numerous diseases and is used as the in vitro model system here. This paper describes the process of collection, purification of small RNA and library generation to sequence extracellular miRNAs. ExRNAs from culture media differ from cellular RNA by being low RNA input samples, which calls for optimized procedures. This protocol provides a comprehensive guide to small exRNA sequencing from culture media, showing quality control checkpoints at each step during exRNA purification and sequencing.

This protocol demonstrates how to purify extracellular microRNAs from cell culture media for small RNA library construction and next generation sequencing. Various quality control checkpoints are described to allow readers to understand what to expect when working with low input samples like exRNAs.

Extracellular and circulating RNAs (exRNA) are produced by many cell types of the body and exist in numerous bodily fluids such as saliva, plasma, serum, ...

Isolation of Papillary and Reticular Fibroblasts from Human Skin by Fluorescence-activated Cell Sorting

Fibroblasts are a highly heterogeneous cell population implicated in the pathogenesis of many human diseases. In human skin dermis, fibroblasts have traditionally been attributed to the superficial papillary or lower reticular dermis according to their histological localization. In mouse dermis, papillary and reticular fibroblasts originate from two different lineages with diverging functions regarding physiological and pathological processes and a distinct cell surface marker expression profile by which they can be distinguished.
Importantly, evidence from explant cultures from superficial and lower dermal layers suggest that at least two functionally distinct dermal fibroblasts lineages exist in human skin dermis as well. However, unlike for mouse skin, cell surface markers enabling the discrimination of different fibroblast subsets have not yet been established for human skin. We developed a novel protocol for the isolation of human papillary and reticular fibroblast populations via fluorescence-activated cell sorting (FACS) using the two cell surface markers Fibroblast Activation Protein (FAP) and Thymocyte antigen 1 (Thy1)/CD90. This method enables the isolation of pure fibroblast subsets without in vitro manipulation, which was shown to affect gene expression, thus permitting accurate functional analysis of human dermal fibroblast subsets in regard to tissue homeostasis or disease pathology.

This manuscript describes a FACS-based protocol for isolation of papillary and reticular fibroblasts from human skin. It circumvents in vitro culture which was inevitable with the commonly used isolation protocol via explant cultures. The emanating fibroblast subsets are functionally distinct and display differential gene expression and localization within the dermis.

Fibroblasts are a highly heterogeneous cell population implicated in the pathogenesis of many human diseases. In human skin dermis, fibroblasts have ...

Sequencing of mRNA from Whole Blood using Nanopore Sequencing

Sequencing in remote locations and resource-poor settings presents unique challenges. Nanopore sequencing can be successfully used under such conditions, and was deployed to West Africa during the recent Ebola virus epidemic, highlighting this possibility. In addition to its practical advantages (low cost, ease of equipment transport and use), this technology also provides fundamental advantages over second-generation sequencing approaches, particularly the very long read length, ability to directly sequence RNA, and real-time availability of data. Raw read accuracy is lower than with other sequencing platforms, which represents the main limitation of this technology; however, this can be partially mitigated by the high read depth generated. Here, we present a field-compatible protocol for sequencing of the mRNAs encoding for Niemann-Pick C1, which is the cellular receptor for ebolaviruses. This protocol encompasses extraction of RNA from animal blood samples, followed by RT-PCR for target enrichment, barcoding, library preparation, and the sequencing run itself, and can be easily adapted for use with other DNA or RNA targets.

Nanopore sequencing is a novel technology that allows cost-effective sequencing in remote locations and resource-poor settings. Here, we present a protocol for sequencing of mRNAs from whole blood that is compatible with such conditions.

Sequencing in remote locations and resource-poor settings presents unique challenges. Nanopore sequencing can be successfully used under such conditions, ...