Has anybody tried miRNA Arrays from Ambion? - (Mar/23/2006 )

Hi everyone,

we are trying to isolate miRNA with the Ambion mirVana miRNA isolation Kit and to hybridize it to the miRNA Bioarray?
Has anyone done it successfully? Has anyone made experiences with the flashPAGE Fractionator?

Thanks
Chris

I'm also using the mirVana kit & found it to be very sucessful in extracting small RNA fragments. How far have you got with it? Are you using Ambions labelling kit aswell? I'd be interested to know how you are getting on with it.

We have done the labelling according to Ambions miRNA Application Guide and also the hybridization.
After scanning the Bioarray we saw signals from the positive control but not from our labelled samples.
So we assume that there was something wrong with the fractionator. have you used it? and how did you check out the isolation of small rnas?

Hi,

Thats interesting! Are you checking the quality of your labelled sample before you do the hyb? How do u know the labelling has been sucessful?

Personally i dont believe the FlashPage is very efficient, i'd check the quality of your original isolation on a gel and if you dont have alot of Ribosomal contamination in your sample i'd try running it though the labelling & hybridization protocol without running it though the FlashPage.

Are you using Cy Dyes or Alexa Dyes for labelling?

Hi,

We don´t check the quality because in the protocol they say that one has to use the sample for the hyb 30 min after elution. but its a good idea, how could one do this?

do you use the enrichment protocol for the small RNAs? Because if you do a gel check you have to use a large quantity of sample to see the small ones.
Without the FlashPage they say there would be a competition in the hybridization between the miRNAs and all the larger molecules- do you believe that is a problem?
We are using Cy Dyes.

Yes i know but 30mins is enough time to quickly check your sample ie. You can check if your Cy Dyes have labelled by checking the OD of the sample. Also yes i am doing the enrichment for small RNAs step but the concentration of my RNA is usually quite high & therefore can run a simple RNA gel by using a small amount of sample diluting it in nuclease free water. It doesnt have to be that accurate to see the MiRNA fraction all you really want to know is if there are any larger fragment bands or not. I guess its for you to decide if it is worth using the flash page but i'd try & run the hybe without it & see what you get.

How many hybs have you done? Have you got consistantly good hybs each time ie, the +ve control comes up strong & the spots are uniform? what tissue are you using, are you checking you are extracting abundant amounts of good quality RNA in the first place?

We are just starting with it and have done only two hybridizations. The first time we saw only the positive control and the second time nothing. in the second hyb we put the positive control before the FlasgPage to our samples and thats why we think the Page doesn´t work.
But our starting rna has got a very good quality, ratio(260/290) was 2.0 and the bioanylyzer was ok, too (Rin was between 9-10). We are using murine liver and HepG2 cells.
How do you messure the OD (260nm like for RNA) and what can one expect?

I would like to ask that what amount of small RNA (<200nt) (ug/100ul) would be isolated using mirVana miRNA Isolation Kit (ambion) ?

Hi I had no luck with Ambion arrays. Instead I used uRNA microarrays from www.microRNAworld.com -these worked much better

hi all
we printed slides with the ambion probe set and the are working find, we had problems with the labelling of the small RNA's, (you can also try the slides from combimetrix, we had some good results with these)

http://www.protocol-online.org/forums/inde...showtopic=14891

good luck