I want to characterize the mammospheres using conventional PCR. I have performed the experiment 3 times but every time I have a problem with the results. I need a detailed protocol for the steps starting from the extraction of the RNA, cDNA, conventional PCR. I'll use CD44, CD24, and Oct4 as markers for BCSCs.
I'm guessing that mammospheres are similar to tumor spheroids? If so how many spheres do you use for your RNA extraction? What kit are the using? And how many replicates. If they spheres are the size I think they are you may need 5-10 of them per RNA extraction. Just place the spheres in lysis buffer and follow the protocol or the kit. You can elute the RNA twice in water but adding water to the column first, spin and taking that same elution and applying it to the column again and spin. I like the qiagen RNA easy kit. Next would be to spec your RNA and see how much you have. If you have 10ug or so per ul that should be fine to just use a regular high capacity cDNA kit. Doesn't have to be the super high capacity kits just a regular one. Do the RT for 2 hours and the you can spec the cDNA.
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