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

Podsumowanie

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.

Streszczenie

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.

Wprowadzenie

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 ....

Protokół

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 fractio....

Wyniki

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.......

Dyskusje

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.......

Materiały

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.