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 b...

<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>

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 ...

This protocol is necessary to evaluate the different roles of defined biochemical elements in the nucleus and cytoplasm and to analyze intracellular interactions between these domains within various cell lines. This technique utilizes commonplace lab equipment and reagents to more efficiently separate the cytoplasmic and nuclear fractions. This protocol can do so less expensively and quicker than other protocols.As studies on cytoplasmic and nuclear interactions grow more widespread, the impact of localization on intracellular events can become better understood. The exchange between the cytoplasm and the nucleus can be analyzed, indicating whether certain molecules during these exchanges can lead to cancer development, as studies on nuclear proteins have indicated. The most critical step in the protocol is modifying the concentration of the lysis buffer to a proper concentration.As usage of lysis buffer largely depends on the disruption of cell membranes and there is natural variants in the effectiveness of lysis buffer on different cell lines. To begin, pellet the cells, using 1.5 milliliter tubes via centrifugation for five minutes at 2000 G.Discard the supernatant using an aspirating pipette. Resuspend the pellet in 300 microliters of ice cold lysis buffer.Then, spin the tubes at a speed of around 12, 000 G at four degrees Celsius for two minutes. Carefully remove the supernatant and place it in a new 1.5 milliliter tube. The remaining pellet will be the nuclear fraction.Next, add 500 microliters of ice cold PBS to the pellet and centrifuge at 2000 G for five minutes at room temperature. To confirm the separation of the compartments using qPCR, first, convert the RNA into complimentary DNA using a commercially available kit. Prepare reverse transcriptase master mix.Add template RNA. Incubate reactions in a thermocycler. Proceed for performing quantitative polymerase chain reaction and analysis.Start with diluting the cDNA synthesis with DEPC water to have a final concentration of 20 nanograms per milliliter. Using a PCR master mix, prepare the reaction for each sample using manual instructions. Acquire commercial probes necessary to detect cytoplasmic and nuclear fractionation.Use MALAT1 as the nuclear marker and TUG1 as the cytoplasmic marker. Calculate each component's volume by multiplying each component by three for each of the technical replicates for the individual sample. For each nuclear and cytoplasmic sample, acquire the mixes nuclear fraction with MALAT1, cytoplasmic fraction with MALAT1, nuclear fraction with TUG1, and cytoplasmic fraction with TUG1.Vortex briefly to mix solutions and transfer 20 microliters of the mixture to each well of an optical reaction plate. Cover the plate with a clear adhesive film utilized for qPCR and centrifuge the plate briefly to eliminate air bubbles, and then spin the sample down at 300 times G for five minutes at room temperature. Using design and analysis software, select the standard curve for the cycling parameters.Using qPCR software, select setup run. On the data file properties page, select method and input cycle parameters. After inputting cycle parameters, select the plate tab and input the samples to be run.Select start run. After the run is complete, create a data spreadsheet with the sample name, target name, and quantification cycle. Begin with the MALAT1 samples to calculate the nuclear fraction.Subtract MALAT1 cytoplasmic fraction from the MALAT1 nuclear fraction. Continue with the MALAT1 samples to calculate the cytoplasmic fraction. Subtract MALAT1 nuclear fraction from MALAT1 cytoplasmic fraction and then calculate the two raised to negative delta CQ.Perform the calculations with TUG1 samples.When TUG1 is present as a positive control for cytoplasmic elements, the cytoplasmic fractionation levels are expected to be higher than nuclear fractionation levels. A negative result where levels of cytoplasmic fractionation are observed in the sample with MALAT1. This indicates contamination of RNA isolated from the nuclear fraction, potentially due to lysis concentrations that are too low or too high.MALAT1 and TUG1 exhibited the highest correlation with nuclear and cytoplasmic separation as confirmed through a Western blot and RNA electrophoresis, which confirmed the purity and quality of the samples. Additionally, the purity of nuclear and cytoplasmic extracts can be demonstrated by a Western blot using lamin as a marker for nuclear fraction and alpha-tubulin as a positive marker for the cytoplasmic fraction. No peak was observed in the cytoplasmic RNA electrophoresis, demonstrating high quality and little or no contamination in the cytoplasmic RNA sample.The usage of lysis buffer is critical for proper fractionation and optimizing the lysis buffer concentration to the desired cell line of interest is essential. This technique allows for the targeted study of nuclear and cytoplasmic interaction, which is widely applicable to a variety of research topics and allows for a better understanding of how localization of specific biological elements may correlate to their function.

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Cancer Research

4.1K visualizações.  University of Miami Miller School of Medicine. Este protocolo é necessário para avaliar os diferentes papéis de elementos bioquímicos definidos no núcleo e citoplasma e para analisar as interações intracelulares entre esses domínios dentro de várias linhagens celulares. Esta técnica utiliza equipamentos de laboratório comuns e reagentes para separar de forma mais eficiente as frações citoplasmática e nuclear. Este protocolo pode fazê-lo de forma menos dispendiosa e mais rápida do que outros protocolos.À medida que ...