The overall goal of this protocol is to provide detailed guidance to use the minigene E1A2 for evaluating global mRNA splicing changes. This is a quick, inexpensive, and simple tool for assessing the function of the target protein in alternative splicing regulation without the necessity of special regimes or laboratory conditions. Begin by culturing HEK 293 cells with stable expression of the gene of interest.
Split the cells using 0.25%trypsin EDTA, then plate 300, 000 cells per well in six-well plates and incubate them for 24 hours at 37 degrees Celsius in 5%carbon dioxide. After 24 hours, check the confluence, and transfect HEK 293 stable cells only when 70 to 80%confluent. Carefully remove the cell culture medium with a pipette, then add two milliliters of complete DMEM medium without antibiotics and put the plate back in the incubator.
Prepare a tube with the transfection buffer, then add two micrograms of pMTE1A DNA, vortex, and add two microliters of transfection reagent. Vortex again and incubate for 10 minutes at room temperature. Remove the plates from the incubator and carefully add the transfection mixture drop-wise.
Six hours after the transfection, add tetracycline to HEK 293 stable cells for Nek4 expression induction. 24 hours after the transfection, verify cell morphology and transfection efficiency using a fluorescent microscope. Use a pipette to remove the cell culture medium, then add cell culture medium with the chemotherapeutics.
Incubate the cells for another 24 hours. To collect RNA, discard the cell culture medium, and add 0.5 to 1 milliliter of RNA extraction reagent directly to the well. If wells are very confluent, use one milliliter of RNA extraction reagent to improve RNA quality.
Homogenize the cells with a pipette and transfer them to a 1.5 milliliter centrifuge tube. Proceed immediately to the RNA extraction, or store the samples at minus 80 degrees Celsius. Thaw the samples in a fume hood and incubate for five minutes at room temperature.
Add 0.1 to 0.2 milliliters of chloroform and agitate vigorously, then incubate for three minutes at room temperature. Centrifuge for 15 minutes at 12, 000 times G and four degrees Celsius, then collect the upper aqueous phase and transfer it to a new 1.5 milliliter centrifuge tube. Collect around 60%of the total volume, but do not collect the DNA or the organic phase.
Add 0.25 to 0.5 milliliters of isopropanol and agitate the sample by inversion four times. Incubate for 10 minutes at room temperature, then centrifuge at 12, 000 times G for 10 minutes at four degrees Celsius and discard the supernatant. Wash the RNA pellet twice with ethanol, and centrifuge at 7, 500 times G for five minutes, then discard the ethanol.
Remove excess ethanol by inverting the tube on a paper towel, then leave the tube open inside a fume hood to partially dry the pellet for five to 10 minutes. Re-suspend the RNA pellet in 15 microliters of DEPC-treated water. Quantify total RNA and verify RNA quality by measuring absorbance at 230, 260, and 280 nanometers.
To verify total RNA quality, run a 1%agarose gel pretreated with 1.2%of a 2.5%sodium hypochlorite solution for 30 minutes. Perform cDNA synthesis using one to two micrograms of total RNA. Combine RNA, one microliter of oligo d-T, one microliter of DNTP, and nuclease-free water to 12 microliters.
Incubate the reaction in the Thermo Cycler for five minutes at 65 degrees Celsius. Remove samples from the Thermo Cycler, and add four microliters of reverse transcriptase buffer, two microliters of DTT, and one microliter of ribonuclease inhibitor. Incubate at 37 degrees Celsius for two minutes.
Add one microliter of thermo-stable reverse transcriptase, and incubate at 37 degrees Celsius for 50 minutes. Inactivate the enzyme at 70 degrees Celsius for 15 minutes. Perform PCR as described in the text manuscript, then load 20 to 25 microliters of the PCR product on a 3%agarose gel containing nucleic acid stain, and run at 100 volts for approximately one hour.
Photograph the gel, avoiding any band saturation, and quantify the bands using an image processing software. The bands at 631, 493, and 156 base pairs correspond to the 13S, 12S, and 9S isoforms respectively. From the software's File menu, open the image file and convert it to gray scale.
Adjust Brightness and Contrast, then remove outlier noise if necessary. Draw a rectangle around the first lane with the rectangle selection tool, and select it through the Analyze, Gels, and Select First Lane command, or by using the keyboard shortcut. Use the mouse to click and hold in the middle of the rectangle on the first lane, and drag it over to the next lane.
Go to Analyze, Gels, and Select Next Lane. Repeat this step for all remaining lanes. After all the lanes are highlighted and numbered, go to Analyze, Gels, and Plot Lanes to draw a profile plot of each lane.
Use the straight line selection tool to draw a line across the base of each peak, corresponding to each band, leaving out the background noise. After all the peaks from every lane have been closed off, select the wand tool and click inside each peak. Measurements should pop up in the results window for each highlighted peak.
Consider only the 13S, 12S, and 9S peaks corresponding to 631, 493, and 156 base pairs bands. Copy intensity data for a spreadsheet editor, and sum the intensity from all three bands for each sample, then calculate the percentage for each isoform relative to the total. Plot the percentages of each isoform, or the differences in the percentage relative to the untreated samples.
A plasmid containing the minigene from E1A was used to observe the effect on alternative splicing by evaluating the proportion of mRNA from each isoform produced. Basal expression of E1A isoforms variants depended on the cell line and time of E1A expression. The HEK 293 stable cell line, or HEK 293 recombinase containing site, showed a higher expression of 13S in comparison to HeLa cells, but showed similar levels of 13S and 12S isoforms after 48 hours of E1A expression.
Cells exposed to cisplatin showed a shift in five prime S-S splicing selection favoring 12S expression. This effect was observed in HEK 293 stably expressing the Flag empty vector, as well as isoform one of Nek4. Changes in alternative splicing after paclitaxel treatment were very discreet, but the directions of the changes were opposite in Flag and Nek4 expressing cells.
When performing this protocol, it's important to use untransfected and non-reverse transcriptase controls to avoid misinterpretation with endogenous E1A expression, mainly when using HEK 293 cells. After obtaining positive results, the alternative splicing of specific genes related to the chemotherapeutic response can be evaluated. When some effect is observed, this simple protocol can direct these studies for more consistent pathways, where the protein plays a role regulating alternative splicing in chemotherapy response, for example.
