The overall goal of this procedure is to use electroporation to introduce siRNAs into difficult to transfect cells in order to study gene expression. This is accomplished by first determining the amount of awa cell suspension needed using the TC 10. Automated cell counter irna is then added to the cell suspension and cells are electro created.
Using the MXL electroporation system cells are incubated for 24 hours post electroporation. Then IL four is added to induce gene transcription in experimental cells. Finally, RNA is extracted and real-time PCR is used to measure levels of gene expression.
Ultimately, results can be obtained that show the effect of specific siRNAs on gene expression pathways. Hi, I'm Adam McCoy, senior scientist for the gene expression division of Bio Rad Laboratories. Today we'll show you procedure for introducing RNAs in a difficult to transfect cell lines by electroporation and then look at the effects on gene expression.
We use this procedure in our laboratory to analyze gene expression of single genes or study gene pathways by knocking out one gene and looking for downstream effects. So let's get started. Working under a cell culture hood, remove the cells from a culture flask.
The cells used in this protocol are suspension cells. If you are using a adherent cells, you will need to trypsin eyes before removing them. Centrifuge the cells to pellet them them.
After the cells have been spun down, remove the supernatant and resuspend the cells in PBS. To determine the number of live cells, take an aliquot of the cell suspension and add trian blue stain at a ratio of one to one to stain the dead cells. Trian blue is required for live dead cell assessment, but not for total cell count.
Only pipe at the mixture onto the opening of a disposable counting slide and insert it into the TC 10 automated cell counter. The TC 10 will first auto-focus to assure the most accurate count and then we'll begin counting the cells. The number of live and total cells per milliliter in the sample is displayed.
Select view image to display the cells on the view screen. Then using the up or down arrows. Zoom in or out as needed to calculate the amount of cell suspension needed for experiments, select dilution calculator.
Enter the desired concentration and final volume values for this experiment. 16 total samples will be prepared, including duplicates for each treatment. So a total of 3.6 milliliters of a five times 10 of the six cells per milliliter suspension will be needed.
The cell counter will then report the volume of cell suspension needed based on the live cell count and the target values entered. If TRIAM blue was used, the counter will automatically adjust for the one-to-one dilution used. Now under a hood, transfer the calculated volume of cell suspension into a new tube.
Then spin the cell mixtures to pellet the cells while the cells are spinning down. Prepare micro fuge tubes for the electroporation by adding SIR A to each tube. For this experiment, one controls irna two stat six specific siRNAs and unresected controls will be prepared Following the centrifugation, remove the PBS and resuspend the cells in 3.6 milliliters of gene Pulser electroporation buffer transfer 800 microliter aliquots into tubes containing the appropriate IRNA and mix gently.
The cells are now ready for electroporation to ate all of the samples simultaneously. Using the MXL electroporation system transfer 150 microliters of each cell suspension into the appropriate wells of a 96 well electroporation plate. Next, put the plate in the plate chamber of the MXL electroporation system and close the lid.
Select the desired electroporation protocol and press pulse to ate the cells after electroporation is complete, remove the plate from the instrument and transfer the cells to a new tissue culture plate containing prewarm to medium. Next, transfer 150 microliters of non-rated control cells for each sample to the new dish. Then place the cells at 37 degrees Celsius, 5%carbon dioxide overnight.
After 24 hours, use IL four to induce gene transcription in one set of cells and add medium only to the second set. One IL four is added to the cells. Incubate an additional 24 hours at 37 degrees Celsius and 5%carbon dioxide after a total of 48 hours of growth.
There can be large differences in cell density if the experimental treatment affects cell division. So it's a good idea to use the TC 10 automated cell counter to quickly count the cells again based on the cell counts. Transfer one aliquot from each well to a micro fuge tube for RNA extraction and freeze.
A second aliquot for western blotting or other protein analysis. Today only the RNA workflow will be shown. Centrifuge the samples to pellet the cells.
Then remove and discard the SUP natin from each tube. Proceed with the RNA extraction using the orum total RNA kit. Once the RNA has been extracted, immediately proceed with the analysis, keeping the samples on ice.
To verify RNA quality and quantity in one step, use the Experian automated electrophoresis station heat denature the samples and the Experian RNA ladder and place on ice. Add a prepared mixture of the gel and stain to the priming. Well on an RNA chip, set the priming code on the priming station.
Place the chip in the automated priming station and press start. Remove the chip from the priming station and load the chip. Vortex the chip to mix samples with loading buffer.
Place the chip into the electrophoresis station and start the run. Experian software will display results automatically in the form of an electropherogram, A results table that displays RNA concentration ribosomal ratio, and the RNA quality indicator number and a virtual gel, which looks similar to a traditional RNA gel. High quality total RNA samples will typically have an RQI of eight to 10.
After verifying the RNA quality, prepare the CD NA reactions here. The IS script ONEstep R-T-P-C-R mix is used to combine CD NA synthesis and real-time PCR. In one reaction, make a master mix containing all the reaction components except the RNA templates for each primer set.
Then distribute the mix into the wells of a PCR plate. After diluting the RNA to a uniform concentration, add the samples to the appropriate wells and triplicate. Seal the plate, place it into the realtime PCR machine, then select the appropriate protocol and begin the run once the run has completed, use the realtime traces to determine expression levels.
The software calculates normalized gene expression from the data based on relative expression of experimental and reference genes in treated and controlled cells. This figure shows an overview of the IL four signaling pathway binding of IL four to its receptor. Leads to diarization and activation of STAT six, activated STAT six translocates to the nucleus and activates transcription of its target.
Genes such as CCL 17 without IL four induction transcription of CCL 17 will remain at low constitutive levels after irna mediated silencing of the STAT six gene. Treatment with IL four should lead to little or no increase in CCL 17 expression. Induction of CCL 17 by IL four was measured in different conditions by quantitative real-time.
PCR samples grown without IL four had a low constitutive level of expression. Regardless of the sir, A delivered as shown in green and blue control samples responded normally to treatment with IL four with an eight to tenfold induction of CCL 17. However, cells transfected with siRNAs targeting STAT six shown in red and pink had diminished CCL L 17 expression in response to IL four supporting the idea that STAT six plays a role in the IL four induced expression of CCL 17.
We've just shown you how to por siRNAs into a difficult to transfect suspension cell line and how to follow a gene expression in those cells. When doing this procedure, it's important to remember to keep things as consistent as possible across all the samples. So that's it.
Thanks for watching and good luck with your experiments.
