Taqman miRNA Assay Controls - not the usal question - (Feb/13/2007 )
So currently I am using Applied Biosystems' Taqman qRT-PCR miRNA assays to quantify miRNA in different mouse cell lines.
The problem arises in that they don't have a 5S or U6 endogenous controls.
They have about 4 different snoRNAs available as endogenous controls.
My question is: how should I go about testing which of these 4 controls is best for my experiments?
The problem arises in that they don't have a 5S or U6 endogenous controls.
They have about 4 different snoRNAs available as endogenous controls.
My question is: how should I go about testing which of these 4 controls is best for my experiments?
Hi
It is really a problem I think that the best is 18S RNA.
The problem arises in that they don't have a 5S or U6 endogenous controls.
They have about 4 different snoRNAs available as endogenous controls.
My question is: how should I go about testing which of these 4 controls is best for my experiments?
Hi
It is really a problem I think that the best is 18S RNA.
They do have RNU6B assays, I have used them. They are designed for human, but tech support told me that they work for some other species, including mouse.
Dave
The problem arises in that they don't have a 5S or U6 endogenous controls.
They have about 4 different snoRNAs available as endogenous controls.
My question is: how should I go about testing which of these 4 controls is best for my experiments?
Hi
It is really a problem I think that the best is 18S RNA.
They do have RNU6B assays, I have used them. They are designed for human, but tech support told me that they work for some other species, including mouse.
Dave
Yes, this is true. I have also used them. But be aware that this assay is for U6b not U6. U6b is a ~45nt long RNA. In my cell line it is found at very low quantities. Whereas, U6 (~106nt) is found at very large quantities (quantified by NB). This also brings up the question: is U6b still reliable as a endogenous control? *At least for my cells, I am not sure...
I have talked to Applied Biosystems and they told me that the way they test their Control assays is they load the "same amount" of RNA for different tissues into different potential endogenous control assays. And then they look as to which control varies the least.
I tested this way, but my method of quantifying RNA was simply Absorbance Readings. This brings alot of error into the equation.
Now I have decided to quantify the RNA by some other method. I plan on using Quant-It RNA kit from Invitrogen which is specific for RNA (i.e. no signal from any DNA or protein that might be in the RNA extraction).
I also plan on using low retention tubes and pipet tips at every step involving nucleic acid transfer. Hopefully, I will get an answer...
I am still unsure if I should also run a NB with the same amounts of RNA and quantify the U6 signal.
Any critiques to this approach?
miRNA in total RNA (not small size RNA) purified by Trizol can be analyzed by using following kits, you can chose U6, 5S even 18S rRNA as reference.
http://www.invitrogen.com/content.cfm?pageid=11568
http://www.systembio.com/QuantiMir.htm
Thanks for bringing this to my attention, I was not aware of this. I think for real time, even if you have low quantities of U6B it should be fine, detection level is really quite good for these assay, right?