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

Summary

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.

Abstract

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.

Introduction

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 cell^4,5. 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.

Protocol

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

1. Prepare 0.25x lysis buffer by mixing the following components provided in Table 1A.
   ​NOTE: Samples require approximately 300 μL of 0.25x lysis buffer. Recommended volume = number of samples x 300 μL.

2. Separation of nuclear and cytoplasmic fractions

1. 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.
2. Resuspend the pellet in 300 μL 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.
3. Spin the tubes at the highest speed (12,000 x g) at 4 °C for 2 min.
4. After centrifugation, carefully remove the supernatant and place it in a new 1.5 mL tube. This will be the cytoplasmic fraction. Approximately 300 μL of the cytoplasmic fraction will be attained.
5. The remaining pellet will be the nuclear fraction. Add 500 μL of ice-cold PBS to the pellet and centrifuge at 2,000 x g for 5 min at room temperature.
   ​NOTE: During this step, excess DNA gets removed.

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).

1. Separate the cytoplasmic RNA samples and the nuclear cytoplasmic samples (as the fractionation steps vary between them).
   1. To separate the cytoplasmic fraction, add 1,050 µL of 3.5x RLT lysis buffer (see Table of Materials) and vortex briefly. Then, add 750 μL 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.
   2. To separate the nuclear fraction, add 600 μL of 3.5x lysis buffer and vortex. Then, add 600 μL of 70% ethanol.
2. 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.
   1. 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.
   2. Add 350 μL of washing buffer from a commercially available purification kit (RW1 buffer, see Table of Materials), spin again, and discard the flow through.
   3. Add DNAse solution (1U/uL) for 15 min at room temperature. Then, add 350 μL of washing buffer, spin again, and discard the flow through.
   4. Add 500 μL of mild washing buffer (RPE, see Table of Materials), spin again, and discard the flow through. Add 500 μL of mild washing buffer, spin again, but only for 2 min.
   5. Move the column to a new 1.5 mL microcentrifuge tube and add 30 μL of water to the spin column. Let the column sit for 1 min prior to spinning down.

4. Validation of nuclear-cytoplasmic separation

1. Perform cDNA synthesis
   1. 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's instructions (see Table of Materials).
   2. 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 °C (Table 1B).
2. Perform the quantitative polymerase chain reaction (qPCR) and analysis.
   1. Dilute the cDNA synthesis with DEPC water (see Table of Materials) to have a final concentration of 20 ng/mL.
   2. Using a PCR master mix, prepare the reaction for each sample using manual instructions as listed below.
   3. 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.
   4. Calculate each component's volume by multiplying each component by 3 for each of the technical replicates for the individual sample.
   5. 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).
   6. Vortex briefly to mix solutions and transfer 20 μL of the mixture to each well of an optical reaction plate (see Table of Materials).
   7. 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.
   8. Using design and analysis software (see Table of Materials), select the standard curve for the cycling parameters (Table 2B).
   9. Using qPCR software, select Set up run (Supplementary Figure 1).
   10. On the Data File Properties page, select Method and input cycle parameters from Table 2B (Supplementary Figure 2).
   11. 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).
3. Perform data analysis
   1. After the run is complete, create a data spreadsheet with the sample name, target name, and quantification cycle(Cq) (Supplementary Table 1).
   2. Begin with the MALAT1 samples to calculate the nuclear fraction. Subtract MALAT1 cytoplasmic fraction from the MALAT1 nuclear fraction (Table 4).
   3. Then, calculate the ΔCq (2^(-value)) (Table 5).
   4. Continue with the MALAT1 samples to calculate the cytoplasmic fraction, subtract MALAT1 nuclear fraction from MALAT1 cytoplasmic fraction, and then calculate the ΔCq (2^(-value)) (Table 6).
      NOTE: Looking at the ΔCq, 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.
   5. With TUG1 samples, perform the same calculation as above (steps 4.3.2-4.3.4) (Table 7).
      NOTE: Looking at the ΔCq, 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).

Representative Results

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).

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. Please click here to view a larger version of this figure.

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 < 0.0001. Error bars denote standard deviation. Please click here to view a larger version of this figure.

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. Please click here to view a larger version of this figure.

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's purity, while alpha-tubulin was used to demonstrate the purity of the cytoplasmic fraction. Please click here to view a larger version of this figure.

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. Please click here to view a larger version of this figure.

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. Please click here to download this Table.

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. Please click here to download this Table.

Table 3: Sequences of nuclear and cytoplasmic markers. The table includes the sequences of the markers utilized to confirm nuclear and cytoplasmic separation. Please click here to download this Table.

Table 4: Procedure for subtracting MALAT1 nuclear and cytoplasmic fractions. An example of data sheet utilized to subtract MALAT1 nuclear fraction - MALAT1 cytoplasmic fraction. Please click here to download this Table.

Table 5: Procedure for calculating ΔCq (2^(-value). An example of the data sheet utilized to calculate the ΔCq (2^(-value)). Please click here to download this Table.

Table 6: Procedure for calculating cytoplasmic fraction. An example of the data sheet utilized to calculate the cytoplasmic fraction. Please click here to download this Table.

Table 7: Procedure for calculating nuclear fraction. An example of the data sheet utilized to calculate the nuclear fraction. Please click here to download this Table.

Table 8: Procedure for confirming successful fractionation result. An example of data sheet utilized to verify successful fractionation. Please click here to download this Table.

Supplementary Figure 1: Image of qPCR machine design and analysis software. An image demonstrating the operation of the qPCR machine. Please click here to download this File.

Supplementary Figure 2: Image of qPCR "Data file properties". An image demonstrating the procedure for setting up parameters for qPCR run. Please click here to download this File.

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. Please click here to download this File.

Supplementary Table 1: Sample name datasheet. An example of data sheet with the sample name, target name, and Cq value. Please click here to download this File.

Discussion

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.

Materials

Name	Company	Catalog Number	Comments
Agilent Tapestation	Agilent	G2991BA	The Agilent TapeStation system is an automated electrophoresis solution for the sample quality control of DNA and RNA samples.
0.5% Nonidet P-40	Thermo-Fischer	28324	Used in the making of lysis buffer
50 mM Tris-Cl pH 8.0	Thermo-Fischer	15568025	Used in the making of lysis buffer
MALAT1 GE Assay	Thermo-Fischer	Hs00273907_s1	Utilized for confirmation of Nuclear fraction.
MicroAmp Optical 96-Well Reaction Plate with Barcode	Thermo-Fischer	4326659	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.
MicroAmp Optical Adhesive Film	Thermo-Fischer	4311971	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
PBS	Gibco	20012-023	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.
Qiagen RNA Clean Up Kit-Rneasy Mini Kit	Qiagen	74106	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.
QuantStudio 6	Thermo-Fischer	A43180	qPCR software utilized during protocol. Includes Design and Analysis Software for analyzing fractionation samples
RLT Buffer	Qiagen	79216	Lysis Buffer from RNA clean-up kit
RPE Buffer	Qiagen	1018013	Wash Buffer from RNA clean-up kit
RW1 Buffer	Qiagen	1053394	Wash Buffer from RNA clean-up kit
Taqman Gene Expression Assays	Thermo-Fischer	4331182	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.
TaqMan Universal PCR Master Mix	Thermo-Fischer	4305719	TaqMan Universal PCR Master Mix is the ideal reagent solution when you need a master mix for multiple 5' 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.
TUG1 GE Assay	Thermo-Fischer	Hs00215501_m1	Utilized for confirmation of Cytoplasmic fraction.
UltraPure DEPC-Treated Water	Thermo-Fischer	750024	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.