I want to check expression level of a gene of interest in an organism. I am new to real time-PCR. Could one kindly guide me how to proceed. I already have designed primers for amplification of the gene of interest. Do I need to additionally design any probes or SYBR itself will dol? A step by step explanation and guidance would be helpful.
The only thing you need is the primers. Since you are new, you need to keep in mind that RNAs are very sensitive to RNases, so that you need to use RNase -free supplies. In addition, when you extract RNA, you need to avoid Rnase contamination, including wearing a mask (or at least you should not talk) besides using RNase-free supplies. The protocol should be with the kit when you buy. The key for good q-RT-PCR includes good RNA sample (check it before you start is favored), specific primers, reasonable size of PCR products (
Hello, i agree with the previous answer. you have to be very careful during your PCR. Apart from the primers you don't have to worry cause the other solutions are pre-ready for you. however do not forget your negative and positive controls to be sure that your results are not false.
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