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