Еля (yelya) wrote in biology,
Еля
yelya
biology

RT--PCR and microarrays

Guys,

I hope somebody with experience can help me with some basic questions about RT-qPCR and microarrays that I knows next to nothing about.

1) Concerning RT-PCR:

Suppose, you know that for a particular experiment you will need to run 20 RT-qPCR reactions. How much total RNA would you start with? Or, putting it another way - suppose you start your experiments with 50 ng of total RNA, then do the RT reaction, and then use it for qPCR - how many reactions would you be able to run, assuming I don't do any pre-amplification steps after cDNA synthesis?

Also, can you share your experience of isolating total RNA from cells (not tissues) - if, say, you start with 100,000 cells, how much total RNA can you isolate, and what method do you use? Have you ever tried isolating RNA from a very small amount of cells (say, 100 or 1000)? Or used something like "Cell-to-CT" kits? What, in your experience, is the minimum number of cells that can yield reliable data?

2) Concerning microarrays:

Have you done microarray experiments using universal RNA (URNA) as an internal standard? Some papers claim that using URNA provides means to compare microarray data across experimental platforms and labs. So, why isn't everyone using it? What are pros and cons?

Thank you for your help!
(x-posted to _scientists_ and lab_gripes
  • Post a new comment

    Error

    default userpic
  • 3 comments
To isolate total RNA from zebrafish, my lab uses Invitrogen's Trizol protocol. It's really simple and efficient so long as proper sterility and RNase-free procedures are maintained. Even 100 eggs can yield high enough levels of RNA for Northern Blots or qrt-PCR.
Thanks. What is the typical amount of RNA you get from 100 eggs?
I've only done the protocol a couple of times myself and I haven't quantified the yield, but running just a couple microliters on an agarose gel produces very bright and distinct bands, which suggests a high concentration.