I need to verify a microarray data for top 5 genes using Relative quantitative Real time per (using delta delta Ct method) I have no previous knowledge to run the experiment. Could somebody describe what I have to do step by step. 1. I have isolated RNA and designed the primers based on the criteria from my reference (housekeeping genes) What shall I do next?
I kindly suggest to learn the method first. RG is not the place to teach you a method. There are plenty of free resources, papers and books, as well as internet sites, even you-tube videos. Google for "quantitative real-time PCR". Learn. Visit a lab doing real-time PCR (you have an instrument? Is there no experience in your group?). Possibly visit a course in this topic. Read the manuals and guides for the instrument you are using. Then feel free to ask when you might have a specific question or problem.
1. First you need to reverse transcribe your RNA into cDNA using any reverse transcription kits (Qiagen has good RT kits)
2. You are going to need mastermix, PCR grade H2O, primers for the gene of interest and primers for your house keeping gene for the PCR reaction.
3. I have used Roch mastermix and roch 96 well plates to run the PCR reaction as our PCR machine is Roch. Different brands of mastermix come with different protocols
4. You normally need to add 1ul of each of your primers, 10 ul of mastermix, 3 ul of water and 5 ul of your cDNA to get 20 ul reactions. However this may very according to your experimental conditions and protocols.
5. Finally you can load each sample as duplicates so 10 ul in each well of the 96 well plate for every reaction and run the PCR
6. Once you have your Ct values then you can do your calculations as described in the following paper:
http://www.ncbi.nlm.nih.gov/pubmed/11846609
Hope this is useful to you.
Sounds good Miriam. Let me know if there is anything else I can help you with.
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