Silencing of BRCA2 to Identify Novel BRCA2-regulated Biological Functions in Cultured Human Cells

The overall goal of this procedure is to silence B RCA two expression by transfection of SIR A.This is accomplished by first preparing the SIR a transfection reagent complexes. Next, a first round of transfection is carried out by adding complexes dropwise to a monolayer of cells that are at 80%co fluency. Then a second round of SIR a transfection is performed using a higher SIR, A to transfection reagent ratio.

Finally, the cell monolayers washed with ice cold PBS and the cells are lysed at four degree Celsius with a detergent based lysis buffer. Ultimately, Western blotting analysis is used to show B RCA A two silencing at the protein level. Main advantage of this technique over existing methods is that it allows efficient gene silencing as well as optimal detection of high molecular weight proteins like the tumor suppressor B RCA two To begin after growing human epithelial cell mono layers at 37 degrees Celsius and 5%carbon dioxide in a humidified chamber, remove the growth medium and use room temperature PBS to wash the cells.

To detach the cells from the plate, add two milliliters of 0.25%trypsin 0.53 millimolar EDTA solution and incubate for three to five minutes depending on the cell type. Neutralize the trypsin by adding two milliliters of complete growth medium containing 10%FBS before transferring the cells to a 15 milliliter tube. Place the tubes into a swinging bucket rotor and centrifuge at 320 to 330 Gs and four degree Celsius for three minutes.

Use five milliliters of antibiotic free medium to resuspend the pellets before using a burker chamber or automated counting system to count the cells. Then seat approximately 0.5 to one times 10 to the six cells in two milliliters of antibiotic free medium into each well of six well plates and incubate for 24 hours to 80%con fluency. After the incubation, proceed with a first round of transfection by diluting six microliters of lipid-based irna specific transfection reagent in 130 microliters of reduced serum medium.

Dilute three microliters of 10 micromolar S irna in 130 microliters of reduced serum medium. Combine the diluted irna with a diluted transfection reagent and incubate for five to 10 minutes at room temperature. During the incubation, remove the medium from the cells and add two milliliters of fresh medium without antibiotics.

Prewarm at 37 degrees Celsius. Next at 250 microliters of the siRNA transfection reagent complexes to each well of cells in a six well plate. Then incubate the cells in a humidified incubator at 37 degrees Celsius with 5%carbon dioxide.

After 24 hours, dilute five microliters of 10 micromolar irna in 130 microliters of reduced serum medium and proceed with a second round of transfection is just demonstrated. The high concentration of sarna in the second round is essential to get high efficiency of B rca. A two silencing.

Incubate the cells at 37 degrees Celsius for one to two days before analysis to prepare cell lysate for immuno blotting analysis. Prepare fresh lysis buffer according to the recipe shown here. Remove the medium from the cells and add two milliliters per well of ice cold PBS to wash the cells.

Once then completely remove the PBS from the plate and add 200 microliters of ice cold lysis buffer to each. Well gently rotate the plate to uniformly distribute the lysis buffer and incubate on a rotator at four degree Celsius for 15 minutes. Then using a cell scraper.

Collect the cell lysate in a micro centrifuge tube on ice centrifuge at 17, 900 Gs and four degrees Celsius for 25 minutes and collect the supernat to carry out immuno blotting analysis. Prepare the running buffer by diluting 50 milliliters of 20 x tris acetate SDS running buffer with 950 milliliters of distilled water. Prepare the sample as follows.

Add 15 micrograms of total cell lysate. Five microliters of four x sample buffer containing lithium ESAL sulfate pH 8.42 microliters of 0.5 molar DTT and lysis buffer to a total volume of 20 microliters. Denature the samples at 55 degrees Celsius for 10 minutes.

It is crucial to use this temperature as b RCA two protein is thermo sensitive. Next, set up the electrophoretic chamber according to the manufacturer's instructions. Then load the samples in 10 microliters of a high molecular weight restain protein standard on a precast gel and run at 120 volts for about two hours.

In the meantime, prepare the transfer buffer with 50 milliliters of 20 x transfer buffer, 100 milliliters of 100%methanol and 850 milliliters of water. Activate the PVDF membrane by soaking in 100%methanol for five minutes. Rinse with distilled water and equilibrate in transfer buffer for at least five minutes.

Soak the sponges of the transfer apparatus in transfer buffer at four degree Celsius until use. Stop the run before the 55 kilodalton blue marker leaves the precast gel and assemble the sandwich beginning with three sponges soaked in transfer buffer. Then one three mm paper saturated in transfer buffer.

Next, add the gel, then the activated PVDF membrane followed by one buffer soaked 3M grade paper and finally three soaked sponges. Place the stack into the apparatus and transfer the proteins at 350 milliamps for four hours and four degrees Celsius or at 180 milliamps and four degrees Celsius overnight. After the transfer, use TBS to wash the membrane and block for one hour with TBST and 5%non-fat dry milk.

Then replace the buffer with nine milliliters of TBST milk buffer containing 30 microliters of anti BCA two rabbit polyclonal antibody and incubate a four degree Celsius overnight. After using TBST to wash the membrane three times for 10 minutes each incubate the filter for one hour with one microliter of horse radish peroxidase conjugated anti rabbit secondary antibody diluted in 10 milliliters of TBST milk buffer. After washing the membrane three times, perform KEMMA luminescence immuno detection following the manufacturer's instructions.

Visualize the bands on high resolution ECL films to confirm the specificity of B rca. A two silencing. A scrambled irna was used side by side with B RCA two irna as shown here as mentioned earlier, it is important to perform a second round of irna transfection with a high irna to transfection ratio to get a high efficiency of B RCA A two silencing.

The results here show the difference in knockdown efficiency between using a low and a high siRNA to transfection ratio Depending on the cell type. The optima minimum time to get the highest level of gene silencing may vary after the second round of siRNA transfection, for example, as shown in this figure, 24 hours is sufficient for NTHY thyroid cells, but up to 48 hours is needed for efficient B RCA A two knockdown in PNT one A prostate cells b RCA two silencing is quite stable until day seven after the second round of transfection. As mentioned earlier, denaturing the cell lysates at a temperature above 55 degrees Celsius result in up to 50%loss of B RCA A two protein.

In this experiment, the sensitivity of B RCA A two silenced PNT one A cells to the PARP inhibitor rucaparib was examined after a 24 hour treatment with the drug. A time-dependent decrease in cell proliferation was observed in B RCA two depleted PNT one A cells compared to controls. After watching this video, it should have a good understanding how to silence long protein encoding genes by a CRNA and how to efficiently detect their protein product through an optimized immuno blockin procedure besides BRC two.

This matter may be applied to many other challenging large proteins, particularly when they are expressed at very low levels in the cells.