isolation of nuclei from fresh frozen glioma tissues: a method to obtain intact nuclei from glioma tumor samples

Isolation of Nuclei from Fresh Frozen Glioma Tissues: A Method to Obtain Intact Nuclei from Glioma Tumor Samples

Begin by transferring 10 to 60 milligrams of fresh frozen tissue sample to a prechilled Petri dish. Mince or chop the fresh frozen tissue with a razor blade into small pieces on ice. Add 500 microliters of chilled nuclei lysis buffer onto the tissue in the Petri dish.
Then, transfer the mixture to a Douncer. Dounce the tissue pieces with the &#34;loose&#34; pestle for about 20 strokes until friction is reduced. Then, dounce it with the &#39;tight&#39;&#160;pestle for 20 strokes to achieve complete tissue homogenization.
Transfer the homogenate into a prechilled 2-milliliter tube and add 1 milliliter of chilled lysis buffer into the Douncer, rinse it, and add it to the tube. Mix it gently and incubate it on ice for 5 minutes, mixing with a wide-bore pipette tip one to two times during the incubation.
Filter the entire homogenate using a 30-micrometer strainer mesh and collect it into a 15-milliliter Falcon tube. Then, transfer it back into a new prechilled 2-milliliter tube. A single strainer is typically sufficient for the entire homogenate.
Check the sample under a light microscope to verify the removal of large debris and the intactness of the nuclear membrane. Nuclei need not be round and the nuclear membrane should not be distorted. If debris is present, repeat the filtration.
Next, centrifuge the nuclei on a benchtop centrifuge for 5 minutes. Remove the supernatant, leaving behind approximately 50 microliters of the nuclei-containing pellet. Gently resuspend the pellet in another milliliter of nuclei lysis buffer and incubate it for 5 minutes on ice.
Repeat the centrifugation. Then, remove the supernatant without disturbing the pellet. Add 500 milliliters of HB and incubate the sample for 5 minutes without resuspending. Then, resuspend the nuclei in another 1 milliliter of HB.
Centrifuge for another 5 minutes. Then, remove the supernatant. Resuspend nuclei in 200 microlitres of HB and transfer the suspension into a new 2-milliliter tube.

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1. Tissue Dissection and Dissociation
<ol>
	<li>Transfer fresh frozen tissue sample (10&#8211;60 mg) to a pre-chilled Petri dish. Mince/chop fresh frozen tissue with a razor blade to small pieces on ice.</li>
	<li>Add 500 &#956;L of chilled nuclei lysis buffer to a pre-chilled 1.5 mL tube. Place the tissue pieces in the 1.5 mL tube containing the nuclei lysis buffer and transfer to a Douncer (Table of Materials). 
	NOTE: There are two Dounce homogenizer pestles: &#8220;loose&#8221; or &#8220;A&#8221; pestle for initial sample reduction and &#8220;tight&#8221; or &#8220;B&#8221; pestle for complete sample homogenization.</li>
	<li value="3">Dounce the tissue pieces with the &#8220;loose&#8221; pestle for about 20 strokes, until friction is reduced. If debris is present, the sample may be pre-cleared by filtration with a 100 mm strainer.</li>
	<li>Dounce with the &#8220;tight&#8221; pestle for 20 strokes to achieve complete tissue homogenization.</li>
	<li>Transfer the homogenate (about 500 &#956;L) into a pre-chilled 2 mL tube. Add 1 mL of chilled lysis buffer. Mix gently and incubate on ice for 5 min. Gently mix with a wide-bore pipette tip and repeat 1&#8211;2 times during the incubation.</li>
	<li>Filter the entire homogenate using a 30 &#956;m strainer mesh, collect into a 15 mL Falcon tube and transfer back into a new pre-chilled 2 mL tube. A single strainer is typically sufficient for the entire homogenate.</li>
	<li>Check under a light microscope to verify the removal of large debris and the intactness of the nuclear membrane. Nuclei need to be round and the nuclear membrane should not be distorted. If debris is present, nuclei can be re-filtered.</li>
	<li>Centrifuge the nuclei at 500 x g for 5 min at 4 &#176;C on a benchtop centrifuge. Remove the supernatant, leaving behind ~50 &#956;L with a pellet containing the nuclei. Gently resuspend the pellet in another 1 mL of nuclei lysis buffer and incubate for 5 min on ice.</li>
	<li>Centrifuge the nuclei at 500 x g for 5 min at 4&#176;C. Remove the supernatant without disturbing the pellet, add 500 mL of 1x homogenization buffer (HB) (Table 1) and incubate for 5 min without resuspending. Then, resuspend the nuclei in another 1.0 mL of 1x HB.</li>
	<li>Centrifuge the nuclei at 500 x g for 5 min at 4 &#176;C. Remove the supernatant and gently resuspend the nuclei in 200 &#956;L of 1x HB into a new 2.0 mL tube.
	Table 1: Preparation of 1x Homogenization Buffer Unstable Solution (2 mL per sample)
	<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
		<tbody>
			<tr>
				<td colspan="3">1x Homogenization Buffer Unstable Solution (2 mL per sample)</td>
			</tr>
			<tr>
				<td>Reagent</td>
				<td>Final Conc.</td>
				<td>Vol per sample (&#956;L)</td>
			</tr>
			<tr>
				<td>6x Homogenization Buffer Unstable</td>
				<td>1x</td>
				<td>333.33</td>
			</tr>
			<tr>
				<td>1 M Sucrose</td>
				<td>320 mM</td>
				<td>640.00</td>
			</tr>
			<tr>
				<td>50 mM EDTA</td>
				<td>0.1 mM</td>
				<td>4.00</td>
			</tr>
			<tr>
				<td>10% NP40</td>
				<td>0.1%</td>
				<td>20.00</td>
			</tr>
			<tr>
				<td>H2O</td>
				<td></td>
				<td>1006.27</td>
			</tr>
		</tbody>
	</table>
	</li>
</ol>

<table><tbody><tr><td>2-Mercaptoethanol</td><td>Sigma</td><td>&amp;nbsp;M6250</td><td></td></tr><tr><td>CaCl2&amp;nbsp;</td><td>Sigma&amp;nbsp;</td><td>21115-100ML</td><td></td></tr><tr><td>EDTA (0.5 M)</td><td>&amp;nbsp;Thermo Scientific&amp;nbsp;</td><td>R1021</td><td></td></tr><tr><td>Falcon 15 mL Conical Centrifuge Tubes</td><td>&amp;nbsp;Fisher Scientific</td><td>352096</td><td></td></tr><tr><td>MACS SmartStrainers (100 &amp;micro;m)&amp;nbsp;</td><td>Miltenyi Biotec</td><td>&amp;nbsp;130-098-463</td><td></td></tr><tr><td>Mg(Ac)2&amp;nbsp;</td><td>Sigma&amp;nbsp;</td><td>63052-100ML</td><td></td></tr><tr><td>NP-40</td><td>&amp;nbsp;Abcam&amp;nbsp;</td><td>ab142227</td><td></td></tr><tr><td>Nuclei Isolation Kit: Nuclei EZ Prep</td><td>&amp;nbsp;Sigma&amp;nbsp;</td><td>NUC101-1KT</td><td></td></tr><tr><td>Safe lock tubes 1.5 mL&amp;nbsp;</td><td>Eppendorf&amp;nbsp;</td><td>30120086</td><td></td></tr><tr><td>Safe lock tubes 2.0 mL&amp;nbsp;</td><td>Eppendorf&amp;nbsp;</td><td>30120094</td><td></td></tr><tr><td>Sucrose&amp;nbsp;</td><td>Sigma&amp;nbsp;</td><td>S0389</td><td></td></tr><tr><td>Wide Bore pipette tips (1000 &amp;micro;L)&amp;nbsp;</td><td>Themo Fisher Scientific&amp;nbsp;</td><td>2079GPK</td><td></td></tr></tbody></table>

Source: Narayanan, A. et al. <a href="https://www.jove.com/v/61542/nuclei-isolation-from-fresh-frozen-brain-tumors-for-single-nucleus">Nuclei Isolation from Fresh Frozen Brain Tumors for Single-Nucleus RNA-seq and ATAC-seq.</a> J. Vis. Exp. (2020)
This video describes the protocol to isolate single-nuclei from fresh frozen glioma tissue. The isolated single-nuclei can be used as specimens for sequencing techniques such as single-nuclei RNA (snRNA) sequencing and single-nuclei Assay for Transposase-Accessible Chromatin (snATAC) sequencing to obtain deeper insights into inter- and intra-tumor heterogeneity observed in glioma of the central nervous tissues.

Source: Narayanan, A. et al. Nuclei Isolation from Fresh Frozen Brain Tumors for Single-Nucleus RNA-seq and ATAC-seq. J. Vis. Exp. (2020)
This video ...

To isolate nuclei, begin by taking a fresh frozen glioma sample - a tumor that originates in the glial cells of the brain - in a Petri dish.
Place the petri dish on ice to avoid tissue degradation.
Next, mince the tissue sample into small pieces.
Add a pre-chilled lysis buffer to the minced tissue pieces. Transfer the mixture to a Dounce&#160; homogenizer. Using the pestle, homogenize the tissue to disintegrate the cells and initiate lysis.
Now, transfer the homogenate to a microcentrifuge tube containing a fresh lysis buffer.
Mechanically disrupt the homogenate using a wide-bore pipette tip. This step facilitates the release of cytoplasmic organelles from the cells.
Subsequently, using an appropriately sized cell strainer, filter the homogenate into a fresh tube to remove tissue debris and other contaminants.
After filtration, take the filtrate containing various cell organelles.
Centrifuge the filtrate at a low speed to pellet the denser nuclei at the bottom while the other cell organelles remain in the supernatant.
Discard the organelle-containing supernatant. Store the crude nuclei-rich pellet in a suitable storage buffer for further downstream sequencing.

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Isolation of Nuclei from Fresh Frozen Glioma Tissues: A Method to Obtain Intact Nuclei from Glioma Tumor Samples

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Isolation of Nuclei from Fresh Frozen Glioma Tissues: A Method to Obtain Intact Nuclei from Glioma Tumor Samples