How to express a rare microRNA
Posted 19 April 2011 - 10:02 AM
I would be grateful if anyone could help me. I am trying to determine the expression pattern of a microRNA believed to be expressed at low levels in whole mount embryos. I have designed an RNA probe against the pri-miR and have done several in-situs using alkaline phosphatase. Although I always get staining, more often than not this is all washed out when I add ethanol in the final step before mounting. However the colour remained in the first and penultimate times I did the in-situ. At first I thought it might be that the amount of ethanol added is critical and that it had to greatly exceeed by many times the amount of residual PBT. However, I made sure of this the last time I did it but the colour still washed out. Does anyone have any clues as to how I can prevent this?
I have also thought about expressing the mature miR. The only system I have read about is the LNA probes by Exiqon, which are quite expensive. Does anyone know of other mature miR probes available that are cheaper? Are there any that you would recommend?
Posted 19 April 2011 - 08:02 PM
Are you talking about expressing the miRNA or cheaper miR probes? I don't have any idea about cheap probe, but if you want to expression the miRNA as a positive control, you can cloning the pre-miR sequence into an expression vector.
Posted 20 April 2011 - 02:44 AM
Are you talking about expressing the miRNA or cheaper miR probes?
Both. I have already designed an RNA probe against the pri-miR and used it in alkaline phosphatase in-situs, but this is not working too well as I explainined above. I also want to see the expression of the mature miR as this could be different from that of the pri-miR. I know you can't do this by designing conventional in-situ probes as the mature miR sequence is too short. The only company I know of that specifically designs mature miR probes is Exiqon. I was wondering if anyone knew of any others that design mature miR probes.