The overall goal of this lipotransfection protocol is to introduce antisense oligonucleotides into the cells and evaluate the efficacy of these drugs on exon inclusion in SMA cells. This method can help answer key questions in the antisense therapy field. Such as, the efficacy of new antisense oligos to provide treatment for patients.
The main advantage of this technique is that it is fast, easy and sensitive enough for the testing of various antisense oligonucleotide chemistries. Though this method can be used for the transfection of antisense oligos into fibroblasts, it can also be applied to other cell types, such as primary myoblasts and various other cell lines. Demonstrating the procedure will be Aleks, a technician from my laboratory.
First, thaw a 10 micromolar solution of LNA/DNA mixmer on ice. Dilute the mixmer solution in serum-deprived media to the required concentration in a 1.5 milliliter tube. Next, dilute the transfection reagent and add the diluted transfection reagent to the mixmer in a one to one ratio.
It is critical that the cell confluency is around 65 to 80%while doing the transfection. If the confluency is over 80%the transfection efficiency will be reduced. Then, incubate the transfection reagent mixture at room temperature for 10 minutes.
After 10 minutes, pipet 900 microliters of serum-deprived medium with 5%fetal bovine serum to the tube. Do not add the FBS before the incubation with the transfection reagent and the LNA/DNA mixmers, as it also affects the transfection efficiency. Then, aspirate the old medium from the culture plate.
Now, add one milliliter of the medium containing the LNA/DNA mixmer to the culture plate. Then, leave the plate in the incubator at 37 degrees Celsius for 24 to 48 hours. Following incubation, aspirate the medium from the culture plate.
Then, pipet one milliliter of GPC reagent to the cells. Next, wash the wells several times with the GPC reagent. After several washes, collect the cells in a 1.5 milliliter tube and keep the samples on ice.
Next, vortex the tube at high speed for 30 seconds. Then, store the tube at minus 80 degrees Celsius for an hour. Now, thaw the sample at room temperature and quickly vortex it.
Next, add 200 microliters of chloroform to the sample and vigorously shake it for 15 seconds. Then, incubate the tube at room temperature for three minutes. After three minutes, centrifuge the sample at 12, 000 g for 15 minutes at four degrees Celsius.
Once the centrifugation is over, collect the aqueous phase from the top of the centrifuge tube. Then transfer the aqueous phase in a new tube. Next, add 500 microliters of isopropanol and one microliter of eight micrograms per microliter of RNA-grade glycogen to the aqueous phase.
Quickly vortex the sample for 10 seconds and incubate at room temperature for 10 minutes. After the incubation, centrifuge the sample at 12, 000 g for 10 minutes at four degrees Celsius. Decant the supernatent after the run is over.
Add one milliliter of cold 75%ethanol and briefly vortex to wash the obtained pellet. Again, centrifuge the sample at 7500 g for five minutes at four degrees Celsius. Then, expel all the ethanol and dry the pellet at room temperature.
After the pellet has dried up, dissolve it in 30 microliters of DNAse/RNAse free water. Then, incubate the sample at 60 degree Celsius for 10 minutes. Next, read the concentration of the RNA at 260 nanometers in the spectrophotometer and adjust it to 15 nanograms per microliter.
To constitute the RT-PCR master mix, add the two X RT-PCR reaction buffer, one step RT-PCR enzyme mix, forward and reverse primers and RNAse/DNAse free distilled water. Distribute the master mix into 0.2 millimeter PCR tubes and add the total RNA for each respective sample. Then start the PCR reaction.
After the RT-PCR is complete, add five microliters of six X loading dye to the amplified product. Then, load five microliters of the mixture on a 2%agarose gel. Start the electrophoresis unit at 100 volts for 40 minutes.
First, the efficiency of different mixmers are studied. To do this, inclusion of the SMN2 exon 7 and GAPDH levels in SMA fibroblasts transfected with the mixmers are analyzed. The gel image shows inclusion of exon 7 where the top and the lower bands represent the exon 7 included and excluded SMN2 RNA.
The inclusion efficiency is also quantified to be 78 to 98%when transfected with the mixmers one through five. On the other hand, no significant results are obtained when mixmer six to eight are used to transfect the patient SMA fibroblasts in comparison to the control. In the experiment, GAPDH is used as a control.
Next, quantitative PCR is done to analyze the relative expression of the full length SMN2 in comparison to GAPDH. A sharp increase in the quantity of the SMN2 transcript level is observed when transfected with mixmers one to three and number five, in comparison to the control. The SMN protein expression level is also studied, which shows an increase in the SMN protein expression level in the patient fibroblast transfected with mixmers one through five.
A sharp 1.5 to 1.9-fold increase in the SMN protein level is observed. Here, the control used is cofilin. On the contrary, mixmer six to eight do not alter the SMN protein expression level.
After watching this video, you should have a good understanding of how to transfect antisense oligo to fibroblasts and evaluate the efficacy in vitro.
