This protocol demonstrate that combining sensation messenger RNA transaction with impedance based real time measurements of cell migration and invasion facilitate the identification of gene that stimulate tumor cell migration and invasion. The impedance based real time cell analysis system enables quantitative, continuous, and comprehensive monitoring of tumor cell migration upon gene expression changes. Genes that are over expressing tumors and stimulate tumor cell migration can be identified as potential target to inhibit metastasis in cancer therapy.
To begin, add 10 microliters of 10x reaction buffer, 1.5 microliters of PNE one, and 10 micrograms of a plasmid DNA to a micro centrifuge tube to linearized the plasmid DNA with the restriction enzyme. Add nuclease free water to bring the reaction volume to 100 microliters. Mix it by tapping and spinning it down before incubating the reaction mixture at 37 degrees Celsius overnight.
Again, spin down and add five microliters of 10%SDS, and one microliter of 20 milligrams per milliliter of RNA grade proteinase K to the reaction mixture before mixing it and spinning it down. After 30 minutes of incubation at 50 degrees Celsius, add two microliters each of 10x transcription buffer, ATP, GTP, UTP, CTP, and T7, and one microgram of a linearized DNA to a micro centrifuge tube for T7 RNA polymerase mediated RNA synthesis. Add nuclease free water to bring the reaction volume to 20 microliters.
After a spin down, incubate the reaction mixture at 37 degrees Celsius for two hours for RNA synthesis. Next, spin down and add one microliter of DNAs and tap before 15 minutes of incubation at 37 degrees Celsius to remove the template DNA. Begin the lithium chloride precipitation of the RNA by adding 10 microliters of 7.5 molar lithium chloride to the reaction mixture.
After tapping and spinning down, incubate the reaction mixture at minus 20 degrees Celsius for 30 minutes. For capping heat the RNA sample at 65 degrees Celsius for 10 minutes and then place it on ice for cooling. Next, add capping reaction components and mix by tapping.
Spin down and incubate the reaction mixture at 37 degrees Celsius for one hour. For poly-A tailing, spin down the sample and add the poly-A tailing reaction components. Then tap, spin down, and incubate the reaction mixture at 37 degrees Celsius for two hours.
For lithium chloride precipitation and quantification of the synthetic mRNA, add 50 microliters of 7.5 molar lithium chloride and perform the lithium chloride precipitation. Remove an aliquot of ECM gel stock from the freezer and keep it on ice. Dilute the 10 milligrams per liter ECM gel to a working concentration by mixing 990 microliters of DMEM with 10 microliters of ECM gel in a micro centrifuge tube.
Mix by gentle pipetting. Add 60 microliters of diluted ECM gel to each of the 16 wells of the upper chamber of the CIM plate. Place the upper chamber of the CIM plate with the plate cover off on a protective plastic sheet inside a carbon dioxide incubator for approximately four hours to form a gel layer.
For electroporation, add one milliliter of DPBS to the tumor cell suspension in each tube. Tap the cell suspension and spin down three times for 10 seconds. Remove the supernatant using a micro pipette.
Add 110 microliters of resuspension buffer R to the cell pellet to obtain 0.1 million cells in 10 microliters. Add synthetic mRNA to the cell pellet to obtain the 0.2 to 20 nanograms per microliter concentration depending on the desired expression level. Mix and resuspend the cell pellet gently by tapping.
Next, electroporate 10 microliters of the cell suspension with an electroporation system at 1, 350 volts for 10 milliseconds with three pulses each time. Once done, transfer the electroporated cells into a new micro centrifuge tube with 1.1 milliliters of DMEM containing 0.5%FBS. Open the analysis program by double clicking on the real-time cell analysis software icon.
After opening the default experiment pattern setup option, select the option for running three experiments separately. Open each cradle by clicking on the number tab. Next, click the experiment notes tab.
Select the folder and save the data by entering the experiment's name. Click on the layout tab and select four wells at a time to set up the quad duplicate wells for each treatment condition. Enter the sample information and click on apply.
Click on the schedule tab, then add a step to set up the two step mode of the cell impedance measurements. Then, click apply to set up the first step. Click on add a step again and enter 10 minutes for the interval and 48 hours for the duration of migration and invasion.
Then, click on apply to set up the second step. One hour before the cell impedance measurement, add 160 microliters of DMEM containing 10%serum or other chemo attractants to the wells of the lower chamber of the CIM plate. Assemble the upper chamber containing the ECM gel coated wells for invasion or uncoated wells for migration with the lower chamber.
For the cell migration assay, add 50 microliters of low serum medium to the wells of the upper chamber of the CIM plate. Place the CIM plate and the cradle of the system in the CO2 incubator. Click on the message tab.
Once the connection's okay, message is displayed, the CIM plate is ready for the experiment. Pre-incubate the assembled CIM plate in the carbon dioxide incubator for 30 to 60 minutes before the real time cell analysis to acclimate the CIM plate to the culture condition. For the baseline reading, click the start button for each cradle.
When the save as window appears, save the experimental file to perform the baseline reading. For cell seating, remove the CIM plate from the cradle and place it in the biosafety cabinet on the plate holder. Then add 100 microliters of electroporated cells containing 100, 000 cells to the wells of the upper chamber of the CIM plate, and leave the CIM plate under the biosafety cabinet for 30 minutes at room temperature.
Measure the cell impedance by moving the fully assembled CIM plate back to the respective cradle. Begin the cell impedance measurement for the second step by clicking on the cradle start button and on the plot tab. Then click the add all button and select the average and standard deviation boxes to visualize the data in real time.
The electroporation of U 118 mg glioblastoma cells with varying concentrations of synthetic crack one mRNA resulted in a concentration dependent increase in flag tagged crack one protein one day after transfection. 0.2 and two nanograms per microliter mRNA led to an undetectable or modest expression of the exogenous crack one protein. 20 nanograms per microliter mRNA resulted in a higher expression level than the endogenous crack one protein.
The cell migration assay indicated that the electroporation was 0.2 or two nanograms per microliter crack one mRNA did not greatly affect cell migration. However, electroporation with 20 nanograms per microliter crack one mRNA led to a clear stimulation of cell migration with more cells migrating between two and 13 hours revealing that glioblastoma cell migration was stimulated by the increase in the crack one protein level. The electroporation with synthetic crack L mRNA led to a robust expression of flag tagged crack L protein one day after transfection.
The invasion of the control cells slowed down with the increased ECM protein concentration. Crack L over expressing cells showed an ECM gel concentration dependent decrease in cell invasion. The comparison between the control and crack L over expressing cells at different ECM gel concentrations indicated that crack L overexpression generally stimulated cell invasion through the ECM gel layer.
The results also suggested that the two cell populations were differentially affected by increasing ECM gel concentration at different time points. Cells should be resuspended quickly, gently, and completely in the resuspended bubble R people electroporation because long incubation of cells in the resuspended bubble R makes cells unhealthy. Fast and slow migrating tumor cells can be isolated to characterize differences between the two cell populations.
