If your highly expressed gene has a Ct of say 15 before knocking it down, and after knocking it down its Ct reaches 35, (but the Ct of your endogenous control doesn´t change*), then the difference in expression is 2e20, which is about a 1,000,000 fold. Now, to answer your question, since I wouldn´t trust a Ct over 35 for quantitation (Cts higher than 35 could be due to contaminant DNA), the answer depends on the Ct of your gene expressed in your basal conditions. If you were to dilute your original sample 4 times, then its Ct would increase to 25. That is, if your original sample has a Ct of 23 and then you dilute the sample in half and run another qPCR, you´d expect its Ct to be 24.
If you diminish the amount of target in half, the Ct increases in one unit. Since I don´t know how familiar you are with qPCR, I´ll start with a brief primer: the more of your target in the sample, the lower its Ct will be. QPCR is one of the techniques with the highest dynamic range, so you can probably determine differences in expression much higher than 400 fold. A Western blot containing all your fractions probed against both 40S- and 60S-specific antibodies should then give you the answer how the 80S/40S/60S ration changes with and without knock-down (re-do the experiment also with the exact same amount of cells for the non-knock-down condition to better compare the relative ratios). The 80S will of course contain both proteins. You could use for example an antibody against RPS27 (MW: 9.5 kDa) to detect the 40S and an antibody against RPL9 (MW: 22 kDa) to detect the 60S. To visualize this information and to semi-quantitativly analyze the ratios, you should then perform a Western blot against exclusive 40S and 60S marker proteins. However, from your earlier characterization of the non-KD cells you already know exactly which fraction contains the 40S, which one the 60S and which one the 80S. Depending on the sensitivity of your hardware (absorbance measurement etc.) and final amount of cells, you might of course not see peaks. Try to aim for as much 40S, 60S and 80S in single, but different fractions as possible.When you have found a good gradient for your cell type and biological question (high resolution of 80S/40S/60S ), I would suggest to use exactly this gradient for a smaller amount of your knock-down cells (use as much as you can possibly get without spending your entire budget on siRNAs.). Therefore, I would propose to perform this experiment with a sufficient amount of you cells (but importantly no knockdown) and find the best gradient for you. The important point is this: For your exact cell type you will have to find the best gradient. That way, all polysomes that you are not interested in, will be stuck in the last fraction and they don't annoy you. Since you are not interested in polysomes, but the ratios of 80S/40S/60S you will need a gradient that resolves well in the "low" molecular weight range, for example 20%-50% sucrose. If you want to purify mainly polysomes you will need a different gradient compared to when you want to purify only the small ribosomal subunit. Such different gradients allow you to have a different dynamic range and resolution of your fractions. 2 would be the following, but I have to say that I have never tried it myself: Next to high quality input lysates, the most critical part of a sucessful polysome profiling experiment is the choice of 'steepness' your sucrose gradient (i.e. I am currently working on stable transfection (psPAX2 and pMD2G) but this will take multiple days and is not a guarantee.
Knock down how to#
I have not been able to find any small molecule inhibitors for this protein and am a bit at a loss as to how to get this knockdown working, as the cancer cells are vehemently opposing knockdown. None of these efforts have resulted in any knockdown of the protein evident via western blot. I wanted to validate these shRNA in my cells via transient transfection before moving to stable knockdown and have tried 6 hour polyethyleneimine transfection (1DNA:4 PEI), Effectene transfection at 1:10, 1:25, and 1:50 ratios (24 hour incubation), and electroporation collection after 3, 24, and 48 hour incubation, all with parallel scrambled controls. I have attempted to knock down this protein using two different Sigma shRNA (77% knockdown or 99% knockdown documented in A549) but have not had any success. I am trying to knock down a protein that has been documented to be heavily upregulated in oral squamous cell carcinoma (confirmed via western blot).
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