RNA labelling for Microarrays - (Mar/23/2006 )

Hi all,

I am curently trying to work up a protocol to hyb microRNAs to microarrays. I can extract the small RNA fraction with no problem but i'm finding that my arrays aren't hybing very well. I know my RNA is good (ive analysed my samples on a bioanalyser) so therefore the problem must be in the labelling step. I am using Ambions labelling kit & protocols with Cy3 & Cy5 dyes. Has anyone else been using these protocols or used any other RNA labelling kits that they have found to be sucessful?

Cheers.

Hi Mir ,
you here not mention whats quantity of total/mRNA you are using in their labeling experiment .
There is a alot of steps where problems may occurs.
Like if you people using very less quantity of toatal RNA/mRNA then you needed for amplification step........... and in this amplification steps at different levels of RNAse contamination.
So first of all you reduce RNAse contamination and use sfficent amount of RNA.
I think problems will be short out.
In our experiment we are using cDNA labelling.
Some time we facing this types of problems when we using RNA storing for more than 3-5 months.

Im using Ambions MirVana kit & 5ug miRNA per expt. See the MiRNA, siRNA.. thread ive moved the topic to there as i felt it was more relivant.

hi mir,

yeah we also had some probs getting the small RNA's labeled with the mivana labeling kit. we tested a couple of other labeling kits (mirCury; exiqon , label-it inbio, ncode; invitrogen) but we decided to go ahead with small RNA labeling kit from kreatech (chemical labelling system). we got some nice support from them and it works quit good,

good luck
A