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With good technique, you can reliably perform RT-PCR or RT-qPCR on a sample size of about 100 cells. Some of the comments are correct that you will need to compensate for the reduction of the initial cell input with additional PCR cycles. However, each magnitude in reduction of cells corresponds to about 3 extra PCR cycles (2^3 = 8 ~= 10), so your overall ...


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You can consider use of single-cell RT-qPCR kits such as this one if you have the budget for them. (Note: I have not personally used this kit, just putting out an idea) The Ambion® Single Cell-to-CT™ Kit contains a complete validated workflow for gene expression analysis for samples containing 1–10 cells. Each kit contains reagents for sample ...


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The polymerase used in PCR is extremely stable at room temperature. I would expect you to be able to continue your PCR reaction from the cycle it was stopped at without too much trouble. However, it is important to not over cycle your product. This will produce non-specific products. If a clean result is imperative, take a 1-5ul sample from your original ...


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In this case you can run your PCR for another 18 cycles (if your total number of cycles was 30). However, do the initial denaturation for 1-2min. You may run the same program again (full 30 cycles) but you'll just end up wasting time because after a point all the dNTPs and primers would be exhausted and further cycles will have no effect on the concentration ...


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If you already know the basic sequence i.e. the fixed regions in the primer; for e.g. the known nucleotides in the sequence - NGATWGCTSATNGC, then you can implement your algorithm like this: Fix the max length of the primers. Lets say 20nt. Generate all combinations of primers (20nt): that is pretty straightforward. You will have 4N × 2(R+Y+S+W+K+M) × ...



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