Name: preparation of cytoplasmic and nuclear long rnas from primary and cultured cells
Uploaded: 2023-04-07T13:20:00.000+00:00
Duration: 6 min 29 s
Description: Watch this Scientific Journal Video about Preparation of Cytoplasmic and Nuclear Long RNAs from Primary and Cultured Cells at JoVE.com

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 disruption of cell membranes, there is natural variance in the effectiveness of lysis buffer on different cell lines as there are slight differences in cell fragility and membrane permeability between different cell lines12. Starting with a baseline of 1x lysis buffer, the steps in the protocol were performed while gradually decreasing the concentration of the lysis buffer to 0.25x, which warranted more effective results. Since the experiment aims to ultimately isolate a large number of subcellular components, ensuring the concentration of lysis buffer is optimized to the cell line of interest is critical to achieving the highest yield and best results.
In addition to the adjustment of lysis buffer concentrations, it is important to note the fragility of RNA and stress caution during the isolation of RNA. RNA is more vulnerable to degradation than DNA and many other cellular elements, as its 2' hydroxyl group can serve as a binding point for RNases, a family of enzymes that can degrade RNA and lead to poor results. RNases are highly capable of RNA degradation, even when presented in minor amounts13. Since RNases are released from cells during and after lysis steps, caution must be taken to prevent contamination, which can lead to unwanted RNA degradation14. The steps include wearing gloves during the experiment and changing these often to avoid transmitting RNases from the environment or surfaces to samples. In addition, RNase-free solutions should be utilized, and a separate workspace should be used when handling RNA. Finally, since RNase can be very resistant to lysis buffer and denaturation, RNase inhibitors can be used to mitigate their negative effects on RNA isolation15.
While the steps during the protocol produced highly successful results, there are some limitations. To begin, while RNA and cytoplasmic components can be largely isolated from each other, the two cannot be fully separated without jeopardizing the quality of the RNA for study. Due to this, any conclusion based on differences in the activity or function of molecules from these separate regions may be skewed slightly by the inability to derive fully isolated samples. It is also important to note that if the protocol is adapted to other cell molecules, such as proteins, some proteins or other cellular elements may be degraded during the separation of nuclear extracts, leading to a loss of activity and affecting one's results16. It is also important to note that during this protocol, qPCR of the nuclear and cytoplasmic fractions using MALAT1 and TUG1 were performed simply as validation of the purity and quality of the samples. To expand on this idea, the relative proportions of nuclear and cytoplasmic RNA were not analyzed or compared during this protocol, as it was used solely for the purpose of validation. This protocol can be adapted, however, to other uses, such as comparing the relative proportions of nuclear and cytoplasmic fractions, but these values must be normalized to the amount of total RNA for an accurate analysis or comparison. The steps for normalizing the cytoplasmic and nuclear fractions to total RNA levels have been previously described in other protocols11.
However, as previously mentioned, this method utilizes common lab materials to generate high-quality RNA extracts cost-effectively. Also, this method of studying nuclear and cytoplasmic interactions proves to be among the most time-effective procedures. Using RNA purity kits leads to very effective RNA clean-up and helps ensure high levels of RNA isolation from different cellular regions, leading to more effective samples to study. Finally, the adaptability of this protocol presents itself as a key advantage, as modifying the lysis buffer concentration can give access to the study of many different cell lines, and a density separation technique such as centrifugation can lead to the isolation of several different cellular elements. As studies on cytoplasmic and nuclear interactions grow more widespread, the impact of localization on cellular components can become better understood. The exchange between the cytoplasm and nucleus can be analyzed, indicating whether certain molecules during these exchanges can lead to cancer development, as studies on nuclear proteins such as XPO1 have indicated17.

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><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.&amp;nbsp;</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&#039; 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 low. Conversely, when TUG1 is present as a positive control for cytoplasmic elements, then cytoplasmic fractionation levels are expected to be higher than nuclear fractionation levels. This can be seen in Figure 2, an example of a positive result confirming Nuclear and Cytoplasmic RNA separation.
A negative result can be seen in Figure 3, in which 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. This study also trialed other primers, but 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 (Figure 4 and Figure 5).
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 fraction10, as shown in Figure 4. There is a clear expression of lamin solely within the nuclear fraction, with a clear expression of alpha-tubulin within the cytoplasmic fraction, indicating relative purity within each sample.
Due to the tendency of RNA to degrade during the process of qPCR, it is necessary to confirm the quality of the RNA yield using RNA electrophoresis. RNA electrophoresis was performed and demonstrated a high quality of RNA due to the presence of a peak at the 32S rRNA subunit, which is a feature only present in nuclear fractions. The presence of this peak indicates nuclear purity and sufficient fractionation. Conversely, no peak was observed in the cytoplasmic RNA electrophoresis, demonstrating high quality and little or no contamination in the cytoplasmic RNA sample11 (Figure 5).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/64199/64199fig01.jpg" /> 
Figure 1: Schematic of protocol steps. After cells are treated, they are lysed and centrifuged for isolation. Next, RNA isolation is performed, and cDNA is generated before the validation of isolation utilizing qPCR. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64199/64199fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/64199/64199fig02.jpg" /> 
Figure 2: Positive result data. The figure highlights protocol success exhibited by high levels of separation of nuclear and cytoplasmic components. MALAT1, a nuclear RNA primer, exhibited statistically significant higher levels of the nuclear fraction. In TUG1, a cytoplasmic RNA primer, statistically significant higher levels of cytoplasmic fraction were observed. High statistical significance was observed in both samples via t-test. ****p &#60; 0.0001. Error bars denote standard deviation. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64199/64199fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/64199/64199fig03.jpg" /> 
Figure 3: Negative result data. The figure highlights an example of negative results using protocol exhibited by high levels of separation of nuclear and cytoplasmic components. MALAT1, a nuclear RNA primer, exhibited cytoplasmic fraction levels caused by nuclear contamination. No differences are observed in statistical significance among the fractions via t-test. Error bars denote standard deviation. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64199/64199fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/64199/64199fig04.jpg" /> 
Figure 4: Western Blot demonstrating the purity of cytoplasmic and nuclear fractions. The figure highlights a western blot of the nuclear and cytoplasmic fractions. Lamin was used to verify the nuclear sample&#39;s purity, while alpha-tubulin was used to demonstrate the purity of the cytoplasmic fraction. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64199/64199fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/64199/64199fig05.jpg" /> 
Figure 5: Confirmation of RNA purity of Cytoplasmic and Nuclear Fractions via RNA electrophoresis. (A) RNA electrophoresis of the nuclear fraction. Arrow demonstrates 32S rRNA, which is only observed in nuclear fractions. (B) RNA electrophoresis of the cytoplasmic fraction. The absence of a 32S peak indicates RNA purity of cytoplasmic fraction. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64199/64199fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Table 1: Composition of lysis buffer, reaction mixtures, and samples. (A) Composition of 0.25x lysis buffer. (B) Composition of reverse transcriptase mix utilized during transcription of RNA to cDNA. (C) Composition of nuclear and cytoplasmic samples using a reverse transcriptase kit. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64199/64199_Table 1.xlsx" target="_blank">Please click here to download this Table.</a>
Table 2: Cyclic parameters. (A) The cycling parameters for thermocycler. These parameters were utilized during qPCR for amplification of the target sequence. (B) The cycling parameters for nuclear and cytoplasmic samples. These parameters were utilized during qPCR for amplification of the target sequence. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64199/64199_Table 2_FINAL.xlsx" target="_blank">Please click here to download this Table.</a>
Table 3: Sequences of nuclear and cytoplasmic markers. The table includes the sequences of the markers utilized to confirm nuclear and cytoplasmic separation. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64199/64199_Table 3_FINAL.xlsx" target="_blank">Please click here to download this Table.</a>
Table 4: Procedure for subtracting MALAT1 nuclear and cytoplasmic fractions. An example of data sheet utilized to subtract MALAT1 nuclear fraction - MALAT1 cytoplasmic fraction. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64199/64199_Table 4.xlsx" target="_blank">Please click here to download this Table.</a>
Table 5: Procedure for calculating &#916;Cq (2^(-value). An example of the data sheet utilized to calculate the &#916;Cq (2^(-value)). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64199/64199_Table 5.xlsx" target="_blank">Please click here to download this Table.</a>
Table 6: Procedure for calculating cytoplasmic fraction. An example of the data sheet utilized to calculate the cytoplasmic fraction. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64199/64199_Table 6.xlsx" target="_blank">Please click here to download this Table.</a>
Table 7: Procedure for calculating nuclear fraction. An example of the data sheet utilized to calculate the nuclear fraction. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64199/64199_Table 7.xlsx" target="_blank">Please click here to download this Table.</a>
Table 8: Procedure for confirming successful fractionation result. An example of data sheet utilized to verify successful fractionation. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64199/64199_Table 8.xlsx" target="_blank">Please click here to download this Table.</a>
Supplementary Figure 1: Image of qPCR machine design and analysis software. An image demonstrating the operation of the qPCR machine. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64199/Supplementary Figure 1.jpg" target="_blank">Please click here to download this File.</a>
Supplementary Figure 2: Image of qPCR &#34;Data file properties&#34;. An image demonstrating the procedure for setting up parameters for qPCR run. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64199/Supplementary Figure 2.jpg" target="_blank">Please click here to download this File.</a>
Supplementary Figure 3: Image of loading samples into the qPCR machine. An image demonstrating the procedure for adding samples to the qPCR software for analysis. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64199/Supplementary Figure 3.jpg" target="_blank">Please click here to download this File.</a>
Supplementary Table 1: Sample name datasheet. An example of data sheet with the sample name, target name, and Cq value. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64199/64199_Supplementary Table 1.xlsx" target="_blank">Please click here to download this File.</a>

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 ...

preparation-cytoplasmic-nuclear-long-rnas-from-primary-cultured

Nanyang Technological University

Research

JoVE Journal

Cancer Research

4.7K 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

Isolation of Cognate RNA-protein Complexes from Cells Using Oligonucleotide-directed Elution

Ribonucleoprotein particles direct the biogenesis and post-transcriptional regulation of all mRNAs through distinct combinations of RNA binding proteins. They are composed of position-dependent, cis-acting RNA elements and unique combinations of RNA binding proteins. Defining the composition of a specific RNP is essential to achieving a fundamental understanding of gene regulation. The isolation of a select RNP is akin to finding a needle in a haystack. Here, we demonstrate an approach to isolate RNPs associated at the 5' untranslated region of a select mRNA in asynchronous, transfected cells. This cognate RNP has been demonstrated to be necessary for the translation of select viruses and cellular stress-response genes.
The demonstrated RNA-protein co-precipitation protocol is suitable for the downstream analysis of protein components through proteomic analyses, immunoblots, or suitable biochemical identification assays. This experimental protocol demonstrates that DHX9/RNA helicase A is enriched at the 5' terminus of cognate retroviral RNA and provides preliminary information for the identification of its association with cell stress-associated huR and junD cognate mRNAs.

This manuscript describes an approach to isolate select cognate RNPs formed in eukaryotic cells via a specific oligonucleotide-directed enrichment. We demonstrate the applicability of this approach by isolating a cognate RNP bound to the retroviral 5' untranslated region that is composed of DHX9/RNA helicase A.

Ribonucleoprotein particles direct the biogenesis and post-transcriptional regulation of all mRNAs through distinct combinations of RNA binding proteins. ...

Biochemistry

RNA Pull-down Procedure to Identify RNA Targets of a Long Non-coding RNA

Long non-coding RNA (lncRNA), which are sequences of more than 200 nucleotides without a defined reading frame, belong to the regulatory non-coding RNA&#39;s family. Although their biological functions remain largely unknown, the number of these lncRNAs has steadily increased and it is now estimated that humans may have more than 10,000 such transcripts. Some of these are known to be involved in important regulatory pathways of gene expression which take place at the transcriptional level, but also at different steps of RNA co- and post-transcriptional maturation. In the latter cases, RNAs that are targeted by the lncRNA have to be identified. That&#39;s the reason why it is useful to develop a method enabling the identification of RNAs associated directly or indirectly with a lncRNA of interest.
This protocol, which was inspired by previously published protocols allowing the isolation of a lncRNA together with its associated chromatin sequences, was adapted to permit the isolation of associated RNAs. We determined that two steps are critical for the efficiency of this protocol. The first is the design of specific anti-sense DNA oligonucleotide probes able to hybridize to the lncRNA of interest. To this end, the lncRNA secondary structure was predicted by bioinformatics and anti-sense oligonucleotide probes were designed with a strong affinity for regions that display a low probability of internal base pairing. The second crucial step of the procedure relies on the fixative conditions of the tissue or cultured cells that have to preserve the network between all molecular partners. Coupled with high throughput RNA sequencing, this RNA pull-down protocol can provide the whole RNA interactome of a lncRNA of interest.

This RNA pull-down method allows identifying the RNA targets of a long non-coding RNA (lncRNA). Based on the hybridization of home-made, designed anti-sense DNA oligonucleotide probes specific to this lncRNA in an appropriately fixed tissue or cell line, it efficiently allows the capture of all RNA targets of the lncRNA.

Long non-coding RNA (lncRNA), which are sequences of more than 200 nucleotides without a defined reading frame, belong to the regulatory non-coding ...

Genetics

Transcriptome Analysis of Single Cells

Many gene expression analysis techniques rely on material isolated from heterogeneous populations of cells from tissue homogenates or cells in culture.1,2,3 In the case of the brain, regions such as the hippocampus contain a complex arrangement of different cell types, each with distinct mRNA profiles. The ability to harvest single cells allows for a more in depth investigation into the molecular differences between and within cell populations. We describe a simple and rapid method for harvesting cells for further processing. Pipettes often used in electrophysiology are utilized to isolate (using aspiration) a cell of interest and conveniently deposit it into an Eppendorf tube for further processing with any number of molecular biology techniques. Our protocol can be modified for the harvest of dendrites from cell culture or even individual cells from acute slices.
We also describe the aRNA amplification method as a major downstream application of single cell isolations. This method was developed previously by our lab as an alternative to other gene expression analysis techniques such as reverse-transcription or real-time polymerase chain reaction (PCR).4,5,6,7,8 This technique provides for linear amplification of the polyadenylated RNA beginning with only femtograms of material and resulting in microgram amounts of antisense RNA. The linearly amplified material provides a more accurate estimation than PCR exponential amplification of the relative abundance of components of the transcriptome of the isolated cell. The basic procedure consists of two rounds of amplification. Briefly, a T7 RNA polymerase promoter site is incorporated into double stranded cDNA created from the mRNA transcripts. An overnight in vitro transcription (IVT) reaction is then performed in which T7 RNA polymerase produces many antisense transcripts from the double stranded cDNA. The second round repeats this process but with some technical differences since the starting material is antisense RNA. It is standard to repeat the second round, resulting in three rounds of amplification. Often, the third round in vitro transcription reaction is performed using biotinylated nucleoside triphosphates so that the antisense RNA produced can be hybridized and detected on a microarray.7,8

In this article we describe a simple method for the harvesting of single cells from rat primary neuronal cultures and subsequent transcriptome analysis using aRNA amplification. This approach is generalizable to any cell type.

Many gene expression analysis techniques rely on material isolated from heterogeneous populations of cells from tissue homogenates or cells in ...

Neuroscience

Cell Based Assays of SINEUP Non-coding RNAs That Can Specifically Enhance mRNA Translation

Targeted-protein enhancement is of importance not only for the study of biological processes but also for therapeutic and biotechnological applications. Here, we present a method to selectively up-regulate protein expression of desired genes in cultured cells by means of synthetic antisense non-coding RNAs known as SINEUPs. This positive control of gene expression is at the post-transcriptional level and exerted by an inverted short interspersed nuclear element (SINE) repeat at the 3&#697; end of SINEUPs that comprises its effector domain (ED). SINEUPs can specifically bind to any protein-coding mRNA of choice through its binding domain (BD), a region designed to complement the sequence within the 5&#697; untranslated region (5&#697; UTR) and around the start codon of the mRNA. Target-specific SINEUPs designed in this manner are transfected to cultured cells, and protein and RNA are extracted for downstream analyses, generally 24-48 h post-transfection. SINEUP-induced protein up-regulation is detected by Western-blot analysis and RNA expression is measured using real-time quantitative reverse transcription PCR. We have observed that BD design is critical for achieving optimum SINEUP activity and that testing different BD sizes and positions with respect to the start codon of the target mRNA is recommended. Therefore, we describe here a semi-automated high-throughput imaging method based on fluorescence detection that can be implemented to target mRNA fused with green fluorescent protein (GFP). SINEUPs specifically enhance translation within normal physiological range of the cell, without altering the target transcript level. This method has been successfully employed against a range of endogenous and exogenous targets, in a wide variety of human, mouse, and insect cell lines along with in vivo systems. Moreover, SINEUPs have been reported to increase antibody production and work as an RNA therapeutic against haploinsufficient genes. The versatile and modular nature of SINEUPs makes them a suitable tool for gene-specific translational control.

SINEUPs are synthetic antisense non-coding RNAs, which contain a binding domain (BD) and an effector domain (ED) and up-regulate translation of target mRNA. Here, we describe detection methods for SINEUPs in cultured cell lines, analysis of their translation-promoting activity by Western-blot and a semi-automated high throughput imaging system.

Targeted-protein enhancement is of importance not only for the study of biological processes but also for therapeutic and biotechnological applications. ...

Overexpressing Long Noncoding RNAs Using Gene-activating CRISPR

Long noncoding RNA (lncRNA) biology is a new and exciting field of research, with the number of publications from this field growing exponentially since 2007. These studies have confirmed that lncRNAs are altered in almost all diseases. However, studying the functional roles for lncRNAs in the context of disease remains difficult due to the lack of protein products, tissue-specific expression, low expression levels, complexities in splice forms, and lack of conservation among species. Given the species-specific expression, lncRNA studies are often restricted to human research contexts when studying disease processes. Since lncRNAs function at the molecular level, one way to dissect lncRNA biology is to either remove the lncRNA or overexpress the lncRNA and measure cellular effects. In this article, a written and visualized protocol to overexpress lncRNAs in vitro is presented. As a representative experiment, an lncRNA associated with inflammatory bowel disease, Interferon Gamma Antisense 1 (IFNG-AS1), is shown to be overexpressed in a Jurkat T-cell model. To accomplish this, the activating clustered regularly interspaced short palindromic repeats (CRISPR) technique is used to enable overexpression at the endogenous genomic loci. The activating CRISPR technique targets a set of transcription factors to the transcriptional start site of a gene, enabling a robust overexpression of multiple lncRNA splice forms. This procedure will be broken down into three steps, namely (i) guide RNA (gRNA) design and vector construction, (ii) virus generation and transduction, and (iii) colony screening for overexpression. For this representative experiment, a greater than 20-fold enhancement in IFNG-AS1 in Jurkat T cells was observed.

Traditional cDNA-based overexpression techniques have a limited applicability for the overexpression of long noncoding RNAs due to their multiple splice forms with potential functionality. This review reports a protocol using CRISPR technology to overexpress multiple splice variants of a long noncoding RNA.

Long noncoding RNA (lncRNA) biology is a new and exciting field of research, with the number of publications from this field growing exponentially since ...

Identification of RNAs Engaged in Direct RNA-RNA Interaction with a Long Non-Coding RNA

The growing role attributed nowadays to long non-coding RNAs (lncRNA) in physiology and pathophysiology makes it crucial to characterize their interactome by identifying their molecular partners, DNA, proteins and/or RNAs. The latter can interact with lncRNA through networks involving proteins, but they can also be engaged in direct RNA/RNA interactions. We, therefore, developed an easy-to-use RNA pull-down procedure that allowed identification of RNAs engaged in direct RNA/RNA interaction with a lncRNA using psoralen, a molecule that cross-links only RNA/RNA interactions. Bioinformatics modeling of the lncRNA secondary structure allowed the selection of several specific antisense DNA oligonucleotide probes with a strong affinity for regions displaying a low probability of internal base pairing. Since the specific probes that were designed targeted accessible regions throughout the length of the lncRNA, the RNA-interaction zones could be delineated in the sequence of the lncRNA. When coupled with a high throughput RNA sequencing, this protocol can be used for the whole direct RNA interactome studies of a lncRNA of interest.

An easy-to-use RNA pull-down protocol is designed for the identification of RNAs engaged in direct RNA/RNA interaction with a long non-coding RNA. The protocol uses psoralen as a fixative to cross-link only RNA/RNA interactions and provides the whole direct RNA interactome of a long non-coding RNA when coupled with RNA sequencing.

The growing role attributed nowadays to long non-coding RNAs (lncRNA) in physiology and pathophysiology makes it crucial to characterize their interactome ...

High-Quality Nuclei from iPSC-Derived Hematopoietic Cells for Multiomics Studies

Nuclear Isolation from Cryopreserved In Vitro Derived Blood Cells

Induced pluripotent stem cell (iPSC)-based models are excellent platforms to understand blood development, and iPSC-derived blood cells have translational utility as clinical testing reagents and transfusable cell therapeutics. The advent and expansion of multiomics analysis, including but not limited to single nucleus RNA sequencing (snRNAseq) and Assay for Transposase-Accessible Chromatin sequencing (snATACseq), offers the potential to revolutionize our understanding of cell development. This includes developmental biology using in vitro hematopoietic models. However, it can be technically challenging to isolate intact nuclei from cultured or primary cells. Different cell types often require tailored nuclear preparations depending on cellular rigidity and content. These technical difficulties can limit data quality and act as a barrier to investigators interested in pursuing multiomics studies. Specimen cryopreservation is often necessary due to limitations with cell collection and/or processing, and frozen samples can present additional technical challenges for intact nuclear isolation. In this manuscript, we provide a detailed method to isolate high-quality nuclei from iPSC-derived cells at different stages of in vitro hematopoietic development for use in single-nucleus multiomics workflows. We have focused the method development on the isolation of nuclei from iPSC-derived adherent stromal/endothelial cells and non-adherent hematopoietic progenitor cells, as these represent very different cell types with regard to structural and cellular identity. The described troubleshooting steps limited nuclear clumping and debris, allowing the recovery of nuclei in sufficient quantity and quality for downstream analyses. Similar methods may be adapted to isolate nuclei from other cryopreserved cell types.

Author Spotlight: Advancing In Vitro Blood Cell Production with Single-Cell Multiomics and Functional Genomics

We describe a protocol for extracting high-quality nuclei from cryopreserved induced pluripotent stem cell-derived stromal/endothelial and blood cell types to support single-nucleus next-generation sequencing analyses. Producing high-quality, intact nuclei is imperative for multiomics experiments but can be a barrier to entry in the field for some laboratories.

Induced pluripotent stem cell (iPSC)-based models are excellent platforms to understand blood development, and iPSC-derived blood cells have translational ...

Developmental Biology

Isolation of Adult Spinal Cord Nuclei for Massively Parallel Single-nucleus RNA Sequencing

Probing an individual cell&#39;s gene expression enables the identification of cell type and cell state. Single-cell RNA sequencing has emerged as a powerful tool for studying transcriptional profiles of cells, particularly in heterogeneous tissues such as the central nervous system. However, dissociation methods required for single cell sequencing can lead to experimental changes in the gene expression and cell death. Furthermore, these methods are generally restricted to fresh tissue, thus limiting studies on archival and bio-bank material. Single nucleus RNA sequencing (snRNA-Seq) is an appealing alternative for transcriptional studies, given that it accurately identifies cell types, permits the study of tissue that is frozen or difficult to dissociate, and reduces dissociation-induced transcription. Here, we present a high-throughput protocol for rapid isolation of nuclei for downstream snRNA-Seq. This method enables isolation of nuclei from fresh or frozen spinal cord samples and can be combined with two massively parallel droplet encapsulation platforms.

Here, we present a protocol to rapidly isolate high-quality nuclei from the fresh or frozen tissue for downstream massively parallel RNA sequencing. We include detergent-mechanical and hypotonic-mechanical tissue disruption and cell lysis options, both of which can be used for isolation of nuclei.

Probing an individual cell&#39;s gene expression enables the identification of cell type and cell state. Single-cell RNA sequencing has emerged as a ...

Analysis of RNA Processing Reactions Using Cell Free Systems: 3' End Cleavage of Pre-mRNA Substrates in vitro

The 3&#8217; end of mammalian mRNAs is not formed by abrupt termination of transcription by RNA polymerase II (RNPII). Instead, RNPII synthesizes precursor mRNA beyond the end of mature RNAs, and an active process of endonuclease activity is required at a specific site. Cleavage of the precursor RNA normally occurs 10-30 nt downstream from the consensus polyA site (AAUAAA) after the CA dinucleotides. Proteins from the cleavage complex, a multifactorial protein complex of approximately 800 kDa, accomplish this specific nuclease activity. Specific RNA sequences upstream and downstream of the polyA site control the recruitment of the cleavage complex. Immediately after cleavage, pre-mRNAs are polyadenylated by the polyA polymerase (PAP) to produce mature stable RNA messages.
Processing of the 3&#8217; end of an RNA transcript may be studied using cellular nuclear extracts with specific radiolabeled RNA substrates. In sum, a long 32P-labeled uncleaved precursor RNA is incubated with nuclear extracts in vitro, and cleavage is assessed by gel electrophoresis and autoradiography. When proper cleavage occurs, a shorter 5&#8217; cleaved product is detected and quantified. Here, we describe the cleavage assay in detail using, as an example, the 3&#8217; end processing of HIV-1 mRNAs.

Analysis of RNA Processing Reactions Using Cell Free Systems: 3' End Cleavage of Pre-mRNA Substrates in vitro

RNA polymerase II synthesizes a precursor RNA that extends beyond the 3' end of the mature mRNA. The end of the mature RNA is generated cotranscriptionally, at a site dictated by RNA sequences, via the endonuclease activity of the cleavage complex. Here, we detail the method to study cleavage reactions in vitro.

The 3&#8217; end of mammalian mRNAs is not formed by abrupt termination of transcription by RNA polymerase II (RNPII). Instead, RNPII synthesizes ...

Biology

Nuclei Isolation from Whole Tissue using a Detergent and Enzyme-Free Method

High-throughput transcriptome and epigenome profiling requires preparation of a single cell or single nuclei suspension. Preparation of the suspension with&#160;intact cell or nuclei involves dissociation and permeabilization, steps that can introduce unwanted noise and undesirable damage. Particularly, certain cell-types such as neurons are challenging to dissociate into individual cells. Additionally, permeabilization of the cellular membrane to release nuclei requires optimization by trial-and-error, which can be time consuming, labor intensive and financially nonviable. To enhance the robustness and reproducibility of sample preparation for high-throughput sequencing, we describe a rapid enzyme and detergent-free column-based nuclei isolation method. The protocol enables efficient isolation of nuclei from the entire zebrafish brain within 20 minutes. The isolated nuclei display intact nuclear morphology and low propensity to aggregate. Further, flow cytometry allows nuclei enrichment and clearance of cellular debris for downstream application. The protocol, which should work on soft tissues and cultured cells, provides a simple and accessible method for sample preparation that can be utilized for high-throughput profiling, simplifying the steps required for successful single-nuclei RNA-seq and ATAC-seq experiments.

Single nuclei isolation relies on dissociation and detergent-based permeabilization of the cell membrane, steps that need optimization and are prone to introducing technical artifacts. We demonstrate a detergent and enzyme-free protocol for rapid isolation of intact nuclei directly from whole tissue, yielding nuclei suitable for single-nucleus RNA-seq (snRNA-Seq) or ATAC-seq.

High-throughput transcriptome and epigenome profiling requires preparation of a single cell or single nuclei suspension. Preparation of the suspension ...

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

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Rozdziały w tym wideo

Isolation of Cognate RNA-protein Complexes from Cells Using Oligonucleotide-directed Elution

RNA Pull-down Procedure to Identify RNA Targets of a Long Non-coding RNA

Transcriptome Analysis of Single Cells

Cell Based Assays of SINEUP Non-coding RNAs That Can Specifically Enhance mRNA Translation

Overexpressing Long Noncoding RNAs Using Gene-activating CRISPR

Identification of RNAs Engaged in Direct RNA-RNA Interaction with a Long Non-Coding RNA

Nuclear Isolation from Cryopreserved In Vitro Derived Blood Cells

Isolation of Adult Spinal Cord Nuclei for Massively Parallel Single-nucleus RNA Sequencing

Analysis of RNA Processing Reactions Using Cell Free Systems: 3' End Cleavage of Pre-mRNA Substrates in vitro

Nuclei Isolation from Whole Tissue using a Detergent and Enzyme-Free Method