Supported by grants from the American Society of Hematology, the Robert Wood Johnson Foundation, the Doris Duke Charitable Foundation, the Edward P. Evans Foundation, and the National Cancer Institute (1K08CA230319).

K562 cells are used for the present study. This protocol has been optimized to work for 1 x 106-5 x 106 million cells. However, the procedure can be scaled up for larger cell quantities by increasing the volumes appropriately.
1. Preparation of the 0.25x lysis buffer
<ol>
	<li>Prepare 0.25x lysis buffer by mixing the following components provided in Table 1A. 
	&#8203;NOTE: Samples require approximately 300 &#956;L of 0.25x lysis buffer. Recommended volume = number of samples x 300 &#956;L.</li>
</ol>
2. Separation of nuclear and cytoplasmic fractions
<ol>
	<li>Pellet the cells using 1.5 mL tubes via centrifugation for 5 min at 2000 x g (room temperature). Discard the supernatant using an aspirating pipette. 
	NOTE: K562 cells were utilized during this protocol. However, the protocol can be adapted to various cell lines.</li>
	<li>Resuspend the pellet in 300 &#956;L of ice cold 0.25x lysis buffer. 
	NOTE: Keep on ice for 2 min and rotate the tube every 45 s to ensure proper lysis of the cells.</li>
	<li>Spin the tubes at the highest speed (12,000 x g) at 4 &#176;C for 2 min.</li>
	<li>After centrifugation, carefully remove the supernatant and place it in a new 1.5 mL tube. This will be the cytoplasmic fraction. Approximately 300 &#956;L of the cytoplasmic fraction will be attained.</li>
	<li>The remaining pellet will be the nuclear fraction. Add 500 &#956;L of ice-cold PBS to the pellet and centrifuge at 2,000 x g for 5 min at room temperature. 
	&#8203;NOTE: During this step, excess DNA gets removed.</li>
</ol>
3. RNA clean-up using an RNA purification kit
NOTE: This protocol was achieved utilizing a commercially available RNA purification kit (see Table of Materials).
<ol>
	<li>Separate the cytoplasmic RNA samples and the nuclear cytoplasmic samples (as the fractionation steps vary between them).
	<ol>
		<li>To separate the cytoplasmic fraction, add 1,050 &#181;L of 3.5x RLT lysis buffer (see Table of Materials) and vortex briefly. Then, add 750 &#956;L of 90% ethanol and load it onto the column from the RNA clean-up kit. 
		NOTE: The solution must be well mixed prior to loading onto the column.</li>
		<li>To separate the nuclear fraction, add 600 &#956;L of 3.5x lysis buffer and vortex. Then, add 600 &#956;L of 70% ethanol.</li>
	</ol>
	</li>
	<li>Load both cytoplasmic and nuclear fractions onto RNA columns (see Table of Materials). 
	NOTE: For the remainder of step 3, both the cytoplasmic and nuclear samples follow the same procedure. However, ensure that these samples remain independent of each other.
	<ol>
		<li>Load both cytoplasmic and nuclear fractions onto RNA columns and spin for 30 s at 8,000 x g at room temperature. Discard the excess flow through.</li>
		<li>Add 350 &#956;L of washing buffer from a commercially available purification kit (RW1 buffer, see Table of Materials), spin again, and discard the flow through.</li>
		<li>Add DNAse solution (1U/uL) for 15 min at room temperature. Then, add 350 &#956;L of washing buffer, spin again, and discard the flow through.</li>
		<li>Add 500 &#956;L of mild washing buffer (RPE, see Table of Materials), spin again, and discard the flow through. Add 500 &#956;L of mild washing buffer, spin again, but only for 2 min.</li>
		<li>Move the column to a new 1.5 mL microcentrifuge tube and add 30 &#956;L of water to the spin column. Let the column sit for 1 min prior to spinning down.</li>
	</ol>
	</li>
</ol>
4. Validation of nuclear-cytoplasmic separation
<ol>
	<li>Perform cDNA synthesis
	<ol>
		<li>Once subcellular compartments of RNA have been extracted and purified, confirm the separation of the compartments using qPCR. To do this, first, convert the RNA into cDNA using a commercially available kit following the manufacturer&#39;s instructions (see Table of Materials).</li>
		<li>Prepare reverse transcriptase mastermix. Add template RNA. Incubate reactions in a thermocycler. 
		NOTE: For random hexamers, the cycling parameters used are provided in Table 2A. Use the reverse transcriptase reaction immediately or store at -20 &#176;C (Table 1B).</li>
	</ol>
	</li>
	<li>Perform the quantitative polymerase chain reaction (qPCR) and analysis.
	<ol>
		<li>Dilute the cDNA synthesis with DEPC water (see Table of Materials) to have a final concentration of 20 ng/mL.</li>
		<li>Using a PCR master mix, prepare the reaction for each sample using manual instructions as listed below.</li>
		<li>Acquire commercial probes necessary to detect cytoplasmic and nuclear fractionation. Use MALAT1 as the nuclear marker and TUG1 as the cytoplasmic marker (see Table of Materials). 
		NOTE: Sequences for MALAT1 and TUG1 are provided in Table 3.</li>
		<li>Calculate each component&#39;s volume by multiplying each component by 3 for each of the technical replicates for the individual sample.</li>
		<li>For each nuclear and cytoplasmic sample, acquire the following mixes for each: (a) Nuclear fraction with MALAT1, (b) Cytoplasmic fraction with MALAT1, (c) Nuclear fraction with TUG1, and (d) Cytoplasmic fraction with TUG1 (Table 1C).</li>
		<li>Vortex briefly to mix solutions and transfer 20 &#956;L of the mixture to each well of an optical reaction plate (see Table of Materials).</li>
		<li>Cover the plate with a clear adhesive film utilized for qPCR (see Table of Materials) and centrifuge the plate briefly to eliminate air bubbles, and then spin the sample down at 300 x g for 5 min at room temperature.</li>
		<li>Using design and analysis software (see Table of Materials), select the standard curve for the cycling parameters (Table 2B).</li>
		<li>Using qPCR software, select Set up run (Supplementary Figure 1).</li>
		<li>On the Data File Properties page, select Method and input cycle parameters from Table 2B (Supplementary Figure 2).</li>
		<li>After inputting cycle parameters, select the Plate tab, and input the samples to be run (Supplementary Figure 3). Select Start Run. 
		NOTE: The steps described were performed in a qPCR machine using a commercially available master mix (see Table of Materials).</li>
	</ol>
	</li>
	<li>Perform data analysis
	<ol>
		<li>After the run is complete, create a data spreadsheet with the sample name, target name, and quantification cycle(Cq) (Supplementary Table 1).</li>
		<li>Begin with the MALAT1 samples to calculate the nuclear fraction. Subtract MALAT1 cytoplasmic fraction from the MALAT1 nuclear fraction (Table 4).</li>
		<li>Then, calculate the &#916;Cq (2^(-value)) (Table 5).</li>
		<li>Continue with the MALAT1 samples to calculate the cytoplasmic fraction, subtract MALAT1 nuclear fraction from MALAT1 cytoplasmic fraction, and then calculate the &#916;Cq (2^(-value)) (Table 6). 
		NOTE: Looking at the &#916;Cq, it is observed that the nuclear MALAT1 fraction (green highlight) has a greater MALAT1 marker than the cytoplasm (red highlight), but now confirmation is required to ensure that the cytoplasmic fraction is predominantly cytoplasm and that the nuclear fraction does not contain cytoplasmic contamination.</li>
		<li>With TUG1 samples, perform the same calculation as above (steps 4.3.2-4.3.4) (Table 7). 
		NOTE: Looking at the &#916;Cq, it is observed that the cytoplasmic TUG1 fraction (green highlight) has a greater TUG1 marker than the nuclear fraction (red highlight), thus confirming good separation of cytoplasmic and nuclear fractions (Table 8, Figure 1).</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Conrad, T., Orom, U. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+fractionation+and+isolation+of+chromatin-associated+RNA.">Cellular fractionation and isolation of chromatin-associated RNA.</a> Methods in Molecular Biology. 1468, 1-9 (2017).</li><li>Bashirullah, A., Cooperstock, R. L., Lipshitz, H. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA+localization+in+development.">RNA localization in development.</a> Annual Review of Biochemistry. 67, 335-394 (1998).</li><li>Liu, X., Fagotto, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+method+to+separate+nuclear,+cytosolic,+and+membrane-associated+signaling+molecules+in+cultured+cells.">A method to separate nuclear, cytosolic, and membrane-associated signaling molecules in cultured cells.</a> Science Signaling. 4 (203), (2011).</li><li>Jansen, R. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=mRNA+localization:+message+on+the+move.">mRNA localization: message on the move.</a> Nature Reviews Molecular Cell Biology. 2 (4), 247-256 (2001).</li><li>Carlevaro-Fita, J., Johnson, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global+positioning+system:+Understanding+long+noncoding+RNAs+through+subcellular+localization.">Global positioning system: Understanding long noncoding RNAs through subcellular localization.</a> Molecular Cell. 73 (5), 869-883 (2019).</li><li>Voit, E. O., Martens, H. A., Omholt, S. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=150+years+of+the+mass+action+law.">150 years of the mass action law.</a> PLOS Computational Biology. 11 (1), 1004012 (2015).</li><li>El-Tanani, M., Dakir el, H., Raynor, B., Morgan, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+nuclear+export+in+cancer+and+resistance+to+chemotherapy.">Mechanisms of nuclear export in cancer and resistance to chemotherapy.</a> Cancers (Basel). 8 (3), 35 (2016).</li><li>Turner, J. G., Dawson, J., Sullivan, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nuclear+export+of+proteins+and+drug+resistance+in+cancer).">Nuclear export of proteins and drug resistance in cancer).</a> Biochemical Pharmacology. 83 (8), 1021-1032 (2012).</li><li>Boesenberg-Smith, K. A., Pessarakli, M. M., Wolk, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessment+of+DNA+yield+and+purity:+an+overlooked+detail+of+PCR+troubleshooting.">Assessment of DNA yield and purity: an overlooked detail of PCR troubleshooting.</a> Clinical Microbiology Newsletter. 34 (1), 1-6 (2012).</li><li>Taylor, 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=Altered+nuclear+export+signal+recognition+as+a+driver+of+oncogenesis+altered+nuclear+export+signal+recognition+drives+oncogenesis.">Altered nuclear export signal recognition as a driver of oncogenesis altered nuclear export signal recognition drives oncogenesis.</a> Cancer Discovery. 9 (10), 1452-1467 (2019).</li><li>Ayupe, A. C., 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=Global+analysis+of+biogenesis,+stability+and+sub-cellular+localization+of+lncRNAs+mapping+to+intragenic+regions+of+the+human+genome.">Global analysis of biogenesis, stability and sub-cellular localization of lncRNAs mapping to intragenic regions of the human genome.</a> RNA Biology. 12 (8), 877-892 (2015).</li><li>Ghuysen, J. -. M., Hakenbeck, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Bacterial Cell Wall. , (1994).</li><li>Fersht, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Enzyme Structure and Mechanism. , (1985).</li><li>Green, M. R., Sambrook, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> How to win the battle with RNase. 2019 (2), (2019).</li><li>Blumberg, D. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Creating+a+ribonuclease-free+environment.">Creating a ribonuclease-free environment.</a> Methods in Enzymology. 152, 20-24 (1987).</li><li>Abmayr, S. M., Yao, T., Parmely, T., Workman, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+nuclear+and+cytoplasmic+extracts+from+mammalian+cells.">Preparation of nuclear and cytoplasmic extracts from mammalian cells.</a> Current Protocols in Molecular Biology. , (2006).</li><li>Taylor, 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=Selinexor,+a+first-in-class+XPO1+inhibitor,+is+efficacious+and+tolerable+in+patients+with+myelodysplastic+syndromes+refractory+to+hypomethylating+agents.">Selinexor, a first-in-class XPO1 inhibitor, is efficacious and tolerable in patients with myelodysplastic syndromes refractory to hypomethylating agents.</a> Blood. 132, 233 (2018).</li></ol>

The authors have no conflicts of interest to disclose.

Throughout the protocol, some steps were taken to optimize the elements to be most effective for the cell line of interest. While the steps within the protocol are relatively straightforward, analysis and minor adjustments during critical aspects of the protocol may be necessary. The most critical step in the protocol is modifying the concentration of the lysis buffer to a proper concentration based on the cell line of interest and the cellular target. Since the utilization of lysis buffer largely depends on the disrupti...

Cellular fractionation into subcellular components allows for the isolation and study of defined biochemical domains and aids in determining the localization of specific cellular processes and how this may impact their function1. Isolation of RNA from different intracellular locations can allow for improved accuracy of genetic and biochemical analysis of transcription level events and other interactions between the nucleus and the cytoplasm, which serves as the primary purpose of the current protocol2. This protocol was developed to ensure the isolation of cytoplasmic and nuclear RNAs to determine their respective roles in nuclear export and to understand how the subcellular localization of RNAs in the nucleus and cytoplasm may impact their function in cellular processes. Using materials from a typical laboratory setting, it was possible to achieve nuclear and cytoplasmic fractionation more effectively and less expensively than previously established protocols without jeopardizing the quality of results3.
Additionally, the exchange of molecules between the cytoplasm and the nucleus can be studied directly by separating these regions. More specifically, understanding the transcriptome is essential for understanding development and disease. However, RNAs may be at different maturation levels at any given time and can complicate downstream analysis. This protocol allows for the ability to isolate RNA from nuclear and cytoplasmic subcellular fractions, which can aid in studies of RNA and allow for a better understanding of a particular RNA of interest, such as the localization of non-coding RNAs or analysis of splice junctions within the nucleus.
This protocol has been optimized for isolating cytoplasmic and nuclear long RNAs, including mRNAs, rRNAs, and long non-coding RNAs (lncRNAs), due to the size selectivity of the RNA purification column utilized, which can be modified to isolate other RNAs of interest. Previously, the function of long RNAs, such as mRNAs and lncRNAs, highly depended on their respective localization within the cell4,5. Therefore, the study of the export from the nucleus to subcellular domains has become more targeted toward understanding the role that exporting RNA or other cellular components can have on the cell. lncRNAs serve as a prime example of this, as their translation and subsequent effects rely largely on proximity and interactions with other forms of RNA6. Furthermore, the exchange of cellular elements between nuclear and cytoplasmic regions is linked to resistance mechanisms to various cancer treatments7. The isolation of intracellular compartments has allowed for the development of nuclear export inhibitors, which has lessened the effects of resistance mechanisms to different therapies8.
Following separating nuclear and cytoplasmic RNA, steps are performed to purify the RNA of interest. Since RNA purification kits are commonly found within laboratories and function to purify and isolate long RNAs, they serve the purpose of this protocol well. For RNA purification, generating 260:280 ratios greater than 1.8 is critical to ensure the quality of samples for RNA sequencing or other similar procedures requiring high levels of purity and isolation. Irregular 260:280 values indicate phenol contamination, demonstrating poor isolation and yielding inaccurate results9.
Once RNA purification is complete and 260:280 values are confirmed to be above an acceptable range, qPCR was utilized to validate the isolation results of nuclear and cytoplasmic fractions. In doing so, primers specific to the region of interest were used to demonstrate the nuclear and cytoplasmic fractionation levels. In this protocol, MALAT1 and TUG1 were used as nuclear and cytoplasmic markers, respectively10. Together, they allow for the demonstration of high levels of nuclear fractionation with MALAT1, while cytoplasmic fractionation is expected to be low. Conversely, when TUG1 is used, cytoplasmic fractionation levels are expected to be higher than nuclear fractionation levels.
Utilizing this protocol, it was possible to isolate RNAs based on their position of action within the cell. Due to widespread access to many materials and utilization of only lysis buffer and density-based centrifugation techniques during this experiment, applicability to other RNA types and other cellular components is widespread. This can provide important information by shedding light on location-specific expression events that would otherwise be indistinguishable without separation.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Agilent Tapestation</td>
			<td>Agilent</td>
			<td>G2991BA</td>
			<td>The Agilent TapeStation system is an automated electrophoresis solution for the sample quality control of DNA and RNA samples.&#160;</td>
		</tr>
		<tr>
			<td>0.5% Nonidet P-40</td>
			<td>Thermo-Fischer</td>
			<td>28324</td>
			<td>Used in the making of lysis buffer</td>
		</tr>
		<tr>
			<td>50 mM Tris-Cl pH 8.0</td>
			<td>Thermo-Fischer</td>
			<td>15568025</td>
			<td>Used in the making of lysis buffer</td>
		</tr>
		<tr>
			<td>MALAT1 GE Assay</td>
			<td>Thermo-Fischer</td>
			<td>Hs00273907_s1</td>
			<td>Utilized for confirmation of Nuclear fraction.</td>
		</tr>
		<tr>
			<td>MicroAmp Optical 96-Well Reaction Plate with Barcode</td>
			<td>Thermo-Fischer</td>
			<td>4326659</td>
			<td>The Applied Biosystems MicroAmp Optical 96-Well Reaction Plate with Barcode is optimized to provide unmatched temperature accuracy and uniformity for fast, efficient PCR amplification. This plate, constructed from a single rigid piece of polypropylene in a 96-well format, is compatible with Applied Biosystems 96-Well Real-Time PCR systems and thermal cyclers.</td>
		</tr>
		<tr>
			<td>MicroAmp Optical Adhesive Film</td>
			<td>Thermo-Fischer</td>
			<td>4311971</td>
			<td>The Applied Biosystems MicroAmp Optical Adhesive Film reduces the chance of well-to-well contamination and sample evaporation when applied to a microplate during qPCR</td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Gibco</td>
			<td>20012-023</td>
			<td>Phosphate-buffered saline (PBS) is a balanced salt solution that is used for a variety of cell culture applications, such as washing cells before dissociation, transporting cells or tissue samples, diluting cells for counting, and preparing reagents.</td>
		</tr>
		<tr>
			<td>Qiagen RNA Clean Up Kit-Rneasy Mini Kit</td>
			<td>Qiagen</td>
			<td>74106</td>
			<td>RNA cleanup kits enable efficient RNA cleanup of enzymatic reactions and cleanup of RNA purified by different methods. Includes RW1, RLT, RW1 buffers mentioned throughout protocol.</td>
		</tr>
		<tr>
			<td>QuantStudio 6</td>
			<td>Thermo-Fischer</td>
			<td>A43180</td>
			<td>qPCR software utilized during protocol. Includes Design and Analysis Software for analyzing fractionation samples</td>
		</tr>
		<tr>
			<td>RLT Buffer</td>
			<td>Qiagen</td>
			<td>79216</td>
			<td>Lysis Buffer from RNA clean-up kit</td>
		</tr>
		<tr>
			<td>RPE Buffer</td>
			<td>Qiagen</td>
			<td>1018013</td>
			<td>Wash Buffer from RNA clean-up kit</td>
		</tr>
		<tr>
			<td>RW1 Buffer</td>
			<td>Qiagen</td>
			<td>1053394</td>
			<td>Wash Buffer from RNA clean-up kit</td>
		</tr>
		<tr>
			<td>Taqman Gene Expression Assays</td>
			<td>Thermo-Fischer</td>
			<td>4331182</td>
			<td>Applied Biosystems TaqMan Gene Expression Assays represent the largest collection of predesigned assays in the industry with over 2.8 million assays across 32 eukaryotic species and numerous microbes. TaqMan Gene Expression assays enable you to get results fast with no time wasted optimizing SYBR Green primers and no extra time spent running and analyzing melt curves.</td>
		</tr>
		<tr>
			<td>TaqMan Universal PCR Master Mix</td>
			<td>Thermo-Fischer</td>
			<td>4305719</td>
			<td>TaqMan Universal PCR Master Mix is the ideal reagent solution when you need a master mix for multiple 5&#39; nuclease DNA applications. Applied Biosystems reagents have been validated with TaqMan assays and Applied Biosystems real-time systems to ensure sensitive, accurate, and reliable performance every time.</td>
		</tr>
		<tr>
			<td>TUG1 GE Assay</td>
			<td>Thermo-Fischer</td>
			<td>Hs00215501_m1</td>
			<td>Utilized for confirmation of Cytoplasmic fraction.</td>
		</tr>
		<tr>
			<td>UltraPure DEPC-Treated Water</td>
			<td>Thermo-Fischer</td>
			<td>750024</td>
			<td>UltraPure DEPC-treated Water is suitable for use with RNA. It is prepared by incubating with 0.1% diethylpyrocarbonate (DEPC), and is then autoclaved to remove the DEPC. Sterile filtered.</td>
		</tr>
	</tbody>
</table>

preparation of cytoplasmic and nuclear long rnas from primary and cultured cells

The separation of intracellular components has been a key tool in cellular biology for many years now and has been able to provide useful insight into how their location can impact their function. In particular, the separation of nuclear and cytoplasmic RNA has become important in the context of cancer cells and the quest to find new targets for drugs. Purchasing kits for nuclear-cytoplasmic RNA extraction can be costly when many of the required materials can be found within a typical lab setting. Using the present method, which can replace more expensive kits or other time-consuming processes, only a homemade lysis buffer, a benchtop centrifuge, and RNA isolation purification columns are needed to isolate nuclear and cytoplasmic RNA. Lysis buffer is used to gently lyse the cell's outer membrane without affecting the integrity of the nuclear envelope, allowing for releasing its intracellular components. Then, the nuclei can be isolated by a simple centrifugation step since they possess a higher density than the lysis solution. Centrifugation is utilized to separate these areas based on their density differences to isolate subcellular elements in the nucleus from those in the cytoplasm. Once the centrifugation has isolated the different components, an RNA clean-up kit is utilized to purify the RNA content, and qPCR is performed to validate the separation quality, quantified by the amount of nuclear and cytoplasmic RNA in the different fractions. Statistically significant levels of separation were achieved, illustrating the protocol's effectiveness. In addition, this system can be adapted for the isolation of different types of RNA (total, small RNA, etc.), which allows for targeted studying of cytoplasm-nucleus interactions, and aids in understanding the differences in the function of RNA that reside in the nucleus and cytoplasm.

To ensure nuclear and cytoplasmic isolation had been achieved, a qPCR was performed to validate the results. In doing so, primers specific to the region of interest were utilized to demonstrate the nuclear and cytoplasmic fractionation levels. In this study, MALAT1 and TUG1 were used as nuclear and cytoplasmic primers, respectively. Together, they allow for the demonstration of high levels of nuclear fractionation with MALAT1 as a positive control for nuclear elements, while cytoplasmic fractionation is expected to be lo...

Watch this Scientific Journal Video about Preparation of Cytoplasmic and Nuclear Long RNAs from Primary and Cultured Cells at JoVE.com

Preparation of Cytoplasmic and Nuclear Long RNAs from Primary and Cultured Cells

The present protocol offers an efficient and flexible method to isolate RNA from nuclear and cytoplasmic fractions using cultured cells, and then validate using qPCR. This effectively serves as a replacement for other RNA preparation kits.

The separation of intracellular components has been a key tool in cellular biology for many years now and has been able to provide useful insight into how ...

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Cancer Research

4.6K Views.  University of Miami Miller School of Medicine. The present protocol offers an efficient and flexible method to isolate RNA from nuclear and cytoplasmic fractions using cultured cells, and then validate using qPCR. This effectively serves as a replacement for other RNA preparation kits.

Video: Preparation of Cytoplasmic and Nuclear Long RNAs from Primary and Cultured Cells

Capture and Release of Viable Circulating Tumor Cells from Blood

We demonstrate a method for size based capture of viable circulating tumor cell (CTC) from whole blood, along with the release of these cells from chip for downstream analysis and/or culture. The strategy employs the use of a novel Parylene C membrane slot pore microfilter to capture CTC and a coating of poly (N-iso-propylacrylamide) (PIPAAm) for thermoresponsive viable release of the captured CTC. The capture of live cells is enabled by leveraging the design of a slot pore geometry with specific dimensions to reduce the shear stress typically associated with the filtration process. While the microfilter exhibits a high capture efficiency, the release of these cells is non-trivial. Typically, only a small percentage of cells are released when techniques such as reverse flow or cell scraping are used. The strong adhesion of these epithelial cancer cells to the Parylene C membrane is attributable to non-specific electrostatic interaction. To counteract this effect, we employed the use of PIPAAm coating and exploited its thermal responsive interfacial properties to release the cells from the filter. Blood is first filtered at room temperature. Below 32 &#176;C, PIPAAm is hydrophilic. Thereafter, the filter is placed in either culture media or a buffer maintained at 37 &#176;C, which results in the PIPAAm turning hydrophobic, and subsequently releasing the electrostatically bound cells.

A protocol to utilize a poly(N-iso-propylacrylamide) (PIPAAm) coated microfilter for effective capture and thermoresponsive release of viable circulating tumor cells (CTC) is presented. This method allows capture of CTC from patients&#39; blood and subsequent release of viable CTC for downstream off-chip culture, analyses and characterization.

We demonstrate a method for size based capture of viable circulating tumor cell (CTC) from whole blood, along with the release of these cells from chip ...

Preparation and Metabolic Assay of 3-dimensional Spheroid Co-cultures of Pancreatic Cancer Cells and Fibroblasts

Many cancer types, including pancreatic cancer, have a dense fibrotic stroma that plays an important role in tumor progression and invasion. Activated cancer associated fibroblasts are a key component of the tumor stroma that interact with cancer cells and support their growth and survival. Models that recapitulate the interaction of cancer cells and activated fibroblasts are important tools for studying the stromal biology and for development of antitumor agents. Here, a method is described for the rapid generation of robust 3-dimensional (3D) spheroid co-culture of pancreatic cancer cells and activated pancreatic fibroblasts that can be used for subsequent biological studies. Additionally, described is the use of 3D spheroids in carrying out functional metabolic assays to probe cellular bioenergetics pathways using an extracellular flux analyzer paired with a spheroid microplate. Pancreatic cancer cells (Patu8902) and activated pancreatic fibroblast cells (PS1) were co-cultured and magnetized using a biocompatible nanoparticle assembly. Magnetized cells were rapidly bioprinted using magnetic drives in a 96 well format, in growth media to generate spheroids with a diameter ranging between 400-600 &#181;m within 5-7 days of culture. Functional metabolic assays using Patu8902-PS1 spheroids were then carried out using the extracellular flux technology to probe cellular energetic pathways. The method herein is simple, allows consistent generation of cancer cell-fibroblast spheroid co-cultures and can be potentially adapted to other cancer cell types upon optimization of the current described methodology.

Here, a method is described for the preparation of 3-dimensional (3D) spheroid co-culture of pancreatic cancer cells and fibroblasts, followed by measurement of metabolic functions using an extracellular flux analyzer.

Many cancer types, including pancreatic cancer, have a dense fibrotic stroma that plays an important role in tumor progression and invasion. Activated ...

Preparation of Primary Acute Lymphoblastic Leukemia Cells in Different Cell Cycle Phases by Centrifugal Elutriation

The ability to synchronize cells has been central to advancing our understanding of cell cycle regulation. Common techniques employed include serum deprivation; chemicals which arrest cells at different cell cycle phases; or the use of mitotic shake-off which exploits their reduced adherence. However, all of these have disadvantages. For example, serum starvation works well for normal cells but less well for tumor cells with compromised cell cycle checkpoints due to oncogene activation or tumor suppressor loss. Similarly, chemically-treated cell populations can harbor drug-induced damage and show stress-related alterations. A technique which circumvents these problems is counterflow centrifugal elutriation (CCE), where cells are subjected to two opposing forces, centrifugal force and fluid velocity, which results in the separation of cells on the basis of size and density. Since cells advancing through the cycle typically enlarge, CCE can be used to separate cells into different cell cycle phases. Here we apply this technique to primary acute lymphoblastic leukemia cells. Under optimal conditions, an essentially pure population of cells in G1 phase and a highly enriched population of cells in G2/M phases can be obtained in excellent yield. These cell populations are ideally suited for studying cell cycle-dependent mechanisms of action of anticancer drugs and for other applications. We also show how modifications to the standard procedure can result in suboptimal performance and discuss the limitations of the technique. The detailed methodology presented should facilitate application and exploration of the technique to other types of cells.

This protocol describes the use of centrifugal elutriation to separate primary acute lymphoblastic leukemia cells into different cell cycle phases.

The ability to synchronize cells has been central to advancing our understanding of cell cycle regulation. Common techniques employed include serum ...

PET and MRI Guided Irradiation of a Glioblastoma Rat Model Using a Micro-irradiator

For decades, small animal radiation research was mostly performed using fairly crude experimental setups applying simple single-beam techniques without the ability to target a specific or well-delineated tumor volume. The delivery of radiation was achieved using fixed radiation sources or linear accelerators producing megavoltage (MV) X-rays. These devices are unable to achieve sub-millimeter precision required for small animals. Furthermore, the high doses delivered to healthy surrounding tissue hamper response assessment. To increase the translation between small animal studies and humans, our goal was to mimic the treatment of human glioblastoma in a rat model. To enable a more accurate irradiation in a preclinical setting, recently, precision image-guided small animal radiation research platforms were developed. Similar to human planning systems, treatment planning on these micro-irradiators is based on computed tomography (CT). However, low soft-tissue contrast on CT makes it very challenging to localize targets in certain tissues, such as the brain. Therefore, incorporating magnetic resonance imaging (MRI), which has excellent soft-tissue contrast compared to CT, would enable a more precise delineation of the target for irradiation. In the last decade also biological imaging techniques, such as positron emission tomography (PET) gained interest for radiation therapy treatment guidance. PET enables the visualization of e.g., glucose consumption, amino-acid transport, or hypoxia, present in the tumor. Targeting those highly proliferative or radio-resistant parts of the tumor with a higher dose could give a survival benefit. This hypothesis led to the introduction of the biological tumor volume (BTV), besides the conventional gross target volume (GTV), clinical target volume (CTV), and planned target volume (PTV).
At the preclinical imaging lab of Ghent University, a micro-irradiator, a small animal PET, and a 7 T small animal MRI are available. The goal was to incorporate MRI-guided irradiation and PET-guided sub-volume boosting in a glioblastoma rat model.

In the past, small animal irradiation was usually performed without the ability to target a well-delineated tumor volume. The goal was to mimic the treatment of human glioblastoma in rats. Using a small animal irradiation platform, we performed MRI-guided 3D conformal irradiation with PET-based sub-volume boosting in a preclinical setting.

For decades, small animal radiation research was mostly performed using fairly crude experimental setups applying simple single-beam techniques without ...

Characterization of Tumor Cells Using a Medical Wire for Capturing Circulating Tumor Cells: A 3D Approach Based on Immunofluorescence and DNA FISH

Circulating tumor cells (CTCs) are associated with poor survival in metastatic cancer. Their identification, phenotyping, and genotyping could lead to a better understanding of tumor heterogeneity and thus facilitate the selection of patients for personalized treatment. However, this is hampered because of the rarity of CTCs. We present an innovative approach for sampling a high volume of the patient blood and obtaining information about presence, phenotype, and gene translocation of CTCs. The method combines immunofluorescence staining and DNA fluorescent-in-situ-hybridization (DNA FISH) and is based on a functionalized medical wire. This wire is an innovative device that permits the in vivo isolation of CTCs from a large volume of peripheral blood. The blood volume screened by a 30-min administration of the wire is approximately 1.5-3 L. To demonstrate the feasibility of this approach, epithelial cell adhesion molecule (EpCAM) expression and the chromosomal translocation of the ALK gene were determined in non-small-cell lung cancer (NSCLC) cell lines captured by the functionalized wire and stained with an immuno-DNA FISH approach. Our main challenge was to perform the assay on a 3D structure, the functionalized wire, and to determine immuno-phenotype and FISH signals on this support using a conventional fluorescence microscope. The results obtained indicate that catching CTCs and analyzing their phenotype and chromosomal rearrangement could potentially represent a new companion diagnostic approach and provide an innovative strategy for improving personalized cancer treatments.

We present a new approach to characterize tumor cells. We combined immunofluorescence with DNA fluorescent-in-situ-hybridization to evaluate cells captured by a functionalized medical wire capable of in vivo enriching CTCs directly from patient blood.

Circulating tumor cells (CTCs) are associated with poor survival in metastatic cancer. Their identification, phenotyping, and genotyping could lead to a ...

An Orthotopic Endometrial Cancer Model with Retroperitoneal Lymphadenopathy Made From In Vivo Propagated and Cultured VX2 Cells

Endometrial cancer is the most common gynecologic malignancy in North America and the incidence is rising worldwide. Treatment consists of surgery with or without adjuvant therapy depending on lymph node involvement as determined by lymphadenectomy. Lymphadenectomy is a morbid procedure, which has not been shown to have a therapeutic benefit in many patients, and thus a new method to diagnose lymph node metastases is required. To test novel imaging agents, a reliable model of endometrial cancer with retroperitoneal lymph node metastases is needed. The VX2 endometrial cancer model has been described frequently in the literature; however, significant variation exists with respect to the method of model establishment. Furthermore, no studies have reported on the use of cultured VX2 cells to create this model as only cells propagated in vivo have been previously used. Herein, we present a standardized surgical method and post-operative monitoring method for the establishment of the VX2 endometrial cancer model and report on the first use of cultured VX2 cells to create this model.

This protocol presents a standardized method to grow VX2 cells in culture and to create an orthotopic VX2 model of endometrial cancer with retroperitoneal lymph node metastases in rabbits. Orthotopic endometrial cancer models are important for the pre-clinical study of novel imaging modalities for the diagnosis of lymph node metastases.

Endometrial cancer is the most common gynecologic malignancy in North America and the incidence is rising worldwide. Treatment consists of surgery with or ...

A Practical Guide for the Production and PET/CT Imaging of 68Ga-DOTATATE for Neuroendocrine Tumors in Daily Clinical Practice

Neuroendocrine tumors are a rare form of cancer that arise from neuroendocrine cells and can be present at almost any location throughout the body. Although heterogeneous in presentation, a common denominator among these tumors is the overexpression of somatostatin receptors. 68Ga-DOTATATE is a somatostatin analog labeled with the positron emitter gallium-68 (68Ga). For well-differentiated neuroendocrine tumors, 68Ga-DOTATATE positron emission tomography (PET)/computed tomography (CT) imaging is used for diagnosis, determination of disease burden, and therapy selection.
This protocol details the radiolabeling of 68Ga-DOTATATE, quality control, patient preparation, and subsequent PET/CT imaging. Radiolabeling of 68Ga-DOTATATE is performed with a fully automated labeling module coupled to a germanium-68 (68Ge)/68Ga generator. Quality control of the final product evaluates radiochemical purity with instant thin-layer chromatography and solid-phase chromatography, and pH prior to patient injection. Periodic quality control is performed to determine 68Ge breakthrough, sterility, and (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) content. Patient preparation includes patient instructions, a protocol for 68Ga-DOTATATE during treatment with somatostatin analogs, and intravenous administration of the radiopharmaceutical. For PET/CT imaging, the acquisition and reconstruction settings are described. For each step, radiation safety will be highlighted, as well as time constrictions due to the short half-life of 68Ga.
Fully automated in-house production and quality control of 68Ga-DOTATATE leads to very high success rates (95%) and produces two to four patient dosages per batch, depending on the yield of the generator. In conclusion, 68Ga-DOTATATE PET/CT imaging is a noninvasive and fast method of providing information on the tumor burden of neuroendocrine tumors (NETs) while also assisting in diagnosis and therapy selection.

A Practical Guide for the Production and PET/CT Imaging of 68Ga-DOTATATE for Neuroendocrine Tumors in Daily Clinical Practice

Well-differentiated neuroendocrine tumors overexpress somatostatin receptors which can be utilized for diagnostic imaging with the radiolabeled somatostatin analog 68Ga-DOTATATE. This protocol details the radiolabeling of 68Ga-DOTATATE, quality control, patient preparation, and subsequent PET/CT imaging. Radiation safety and time constrictions due to the short half-life of 68Ga are taken into account.

Neuroendocrine tumors are a rare form of cancer that arise from neuroendocrine cells and can be present at almost any location throughout the body. ...

Extraction of Aqueous Metabolites from Cultured Adherent Cells for Metabolomic Analysis by Capillary Electrophoresis-Mass Spectrometry

Metabolomic analysis is a promising omics approach to not only understand the specific metabolic regulation in cancer cells compared to normal cells but also to identify biomarkers for early-stage cancer detection and prediction of chemotherapy response in cancer patients. Preparation of uniform samples for metabolomic analysis is a critical issue that remains to be addressed. Here, we present an easy and reliable protocol for extracting aqueous metabolites from cultured adherent cells for metabolomic analysis using capillary electrophoresis-mass spectrometry (CE-MS). Aqueous metabolites from cultured cells are analyzed by culturing and washing cells, treating cells with methanol, extracting metabolites, and removing proteins and macromolecules with spin columns for CE-MS analysis. Representative results using lung cancer cell lines treated with diamide, an oxidative reagent, illustrate the clearly observable metabolic shift of cells under oxidative stress. This article would be especially valuable to students and investigators involved in metabolomics research, who are new to harvesting metabolites from cell lines for analysis by CE-MS.

The purpose of this article is to describe a protocol for extraction of aqueous metabolites from cultured adherent cells for metabolomic analysis, particularly, capillary electrophoresis-mass spectrometry.

Metabolomic analysis is a promising omics approach to not only understand the specific metabolic regulation in cancer cells compared to normal cells but ...

Subcellular Fractionation of Primary Chronic Lymphocytic Leukemia Cells to Monitor Nuclear/Cytoplasmic Protein Trafficking

Nuclear export of macromolecules is often deregulated in cancer cells. Tumor suppressor proteins, such as p53, can be rendered inactive due to aberrant cellular localization disrupting their mechanism of action. The survival of chronic lymphocytic leukaemia (CLL) cells, among other cancer cells, is assisted by the deregulation of nuclear to cytoplasmic shuttling, at least in part through deregulation of the transport receptor XPO1 and the constitutive activation of PI3K-mediated signaling pathways. It is essential to understand the role of individual proteins in the context of their intracellular location to gain a deeper understanding of the role of such proteins in the pathobiology of the disease. Furthermore, identifying processes that underlie cell stimulation and the mechanism of action of specific pharmacological inhibitors, in the context of subcellular protein trafficking, will provide a more comprehensive understanding of the mechanism of action. The protocol described here enables the optimization and subsequent efficient generation of nuclear and cytoplasmic fractions from primary chronic lymphocytic leukemia cells. These fractions can be used to determine changes in protein trafficking between the nuclear and cytoplasmic fractions upon cell stimulation and drug treatment. The data can be quantified and presented in parallel with immunofluorescent images, thus providing robust and quantifiable data.

This protocol enables the optimization and subsequent efficient generation of nuclear and cytoplasmic fractions from primary chronic lymphocytic leukemia cells. These samples are used to determine protein localization as well as changes in protein trafficking that take place between the nuclear and cytoplasmic compartments upon cell stimulation and drug treatment.

Nuclear export of macromolecules is often deregulated in cancer cells. Tumor suppressor proteins, such as p53, can be rendered inactive due to aberrant ...

Preparation of Mitochondria from Ovarian Cancer Tissues and Control Ovarian Tissues for Quantitative Proteomics Analysis

Ovarian cancer is a common gynecologic cancer with high mortality but unclear molecular mechanism. Most ovarian cancers are diagnosed in the advanced stage, which seriously hampers therapy. Mitochondrial changes are a hallmark of human ovarian cancers, and mitochondria are the centers of energy metabolism, cell signaling, and oxidative stress. In-depth insights into the changes of the mitochondrial proteome in ovarian cancers compared to control ovarian tissue will benefit in-depth understanding of the molecular mechanisms of ovarian cancer, and the discovery of effective and reliable biomarkers and therapeutic targets. An effective mitochondrial preparation method coupled with an isobaric tag for relative and absolute quantification (iTRAQ) quantitative proteomics are presented here to analyze human ovarian cancer and control mitochondrial proteomes, including differential-speed centrifugation, density gradient centrifugation, quality assessment of mitochondrial samples, protein digestion with trypsin, iTRAQ labeling, strong cation exchange fractionation (SCX), liquid chromatography (LC), tandem mass spectrometry (MS/MS), database analysis, and quantitative analysis of mitochondrial proteins. Many proteins have been successfully identified to maximize the coverage of the human ovarian cancer mitochondrial proteome and to achieve the differentially expressed mitochondrial protein profile in human ovarian cancers.

This article presents a protocol of differential-speed centrifugation in combination with density gradient centrifugation to separate mitochondria from human ovarian cancer tissues and control ovarian tissues for quantitative proteomics analysis, resulting in a high-quality mitochondrial sample and high-throughput and high-reproducibility quantitative proteomics analysis of a human ovarian cancer mitochondrial proteome.

Ovarian cancer is a common gynecologic cancer with high mortality but unclear molecular mechanism. Most ovarian cancers are diagnosed in the advanced ...