The last post i mentioned was about my project. Till date everything has been going on pretty smootly. I had updated the flow chart a few weeks ago and i have completed the soft agar assay, colony assay and the western blotting. The FACS analysis and the immunocytochemistry is still in progress. 

The results from the following 3 categories have been good. The controls for the experiment turned out positive while the samples itself produced certain surprises. 

Just a short recap. Aurora A is increased significantly in certain cancers. Recently, an Aurora-A Interacting Kinase (AIP) is discovered to react inversely to Aurora A. In this project, we aim to knock down AIP and find out if this characteristic alone, is enough to increase he levels of Aurora-A to form malignancy.

As i mentioned before, the project outcome is kinda anticipated, due to the established researches done previously, where Aurora A is exogenously, over-expressed. We knock down AIP of 3 cell lines, HEK, HEK RAS #1 and HEK RAS #3. Both HEK RAS #1 and HEK RAS #3 are RAS mutated, thus more possible to form tumors upon AIP knockdown.

The colony and soft agar assay is ready to be stained after 2-3 weeks of incubation.

1. Discard old media

2. Wash wells in PBS

3. Stain cells using 2ml of 0.05% crystal violet

4. Allow to settle at RT for 15mins

5. Discard stain.

6. Invert the wells to air dry

7. Snap a UV photo.

1. Discard old media

2. Carefully aliquot 2ml of 0.005% of crystal violet. 

3. Allow to settle overnight

4. Discard excess stain

5.  Invert wells to air dry

6. Snap a UV photo

It is anticipated that the likelihood of HEK RAS #1 results should steers towards malignancy, since malignancy is usually promoted in cells where more mutations occur. This result is accurate for the colony assay where many large intensly stained colonies are formed, indicating its aggressive growth. Since such aggressive growth is observed, we should expect to see formation of colonies on the soft agar assay. However this was not the case, infact no colonies showed in any of the samples, including the controls. The results was puzzling and was investigated that most probably the amount of cells seeded in the soft agar assay was too little. (Approx. 6000cells to be seeded in each dish) Since the cells were RAS mutated, the chance of forming tumors (indicated by colonies on the soft agar assay) is higher. Besides the excessive growth on the colony assay acted as evidence for expecting tumors. 

After explaining, it is now clear and obvious that nothing should appear on the soft agar assays for the HEK samples. The colony assay however should show colonies due to its fastedious characteristics. However the staining intensity of the cells would not be as severe as compared to those of the HEK RAS #1.

As for HEK RAS #3, the results will only be reaped next week.

For western blotting, the results of all 3 cells are complied and analyzed. The flims show the anticipated results for all cells. After the transfer of the protein from the gel to the membrane, the membrane is probed 4 times, each with different antibodies. 

AIP, would detect the protein levels of AIP in the cells, this would check to see if the sample cells have sucessfully knockdown the AIP gene.

AIK, would detect the protein levels of Aurora-A. In this case of the knocked down AIP gene cells, Aurora A levels should show an increase

RAS, would detect RAS proteins. This checks the stability of the RAS mutation.

Beta-Tubulin, house keeping gene. Ensures that the cells are mammalian, acts like a control.

As mentioned before, the cells were subjected to different concentrations of siAIPs that would "block" and knockdown the AIP gene. Logically speaking more siAIP, blocks more of the AIP, thus more knocked down, eventually less amount of AIP produced. Therefore 2 samples, 2.5nm siAIP and 20nm siAIP should show slight differences in AIP/AIK protein production.  All 3 cells are able to show slight differences.

When detecting AIP-Ab on the membrane, the results would show very faint bands for 2.5nm, while the sample 20nm band, would be even lighter. And while detecting AIK-Ab, the bands from 2.5nm would be slight lighter in intensity than those from 20nm sample. (since AIP and AIK are inversly proportionate)

Alright. So far these are my results. I have already fixed the cells for FACS analysis, and am going to process the cell cycle of the cells next week. 

Enjoy the last 10weeks(:

Glad