Posted 29 October 2010 - 04:41 PM
Need some help regarding Northern Blot. I am trying to detect a pre-miRNA band in Northern Blot. Although I get amplification for the pre-miRNA in RT-PCR,can't detct the corresponding band in Northern analysis. My positive controls for the transfer and hybridization are working fine. I am loading approaximately 2ug of <200bp RNA isolated using Qiagen mi-RNA enrichment kit.
Thank you for your suggestions.
Posted 29 October 2010 - 04:45 PM
My first thought would be that your < 200 bp pre-miRNA is not being transferred to your membrane. Because of its small site it is quite possibly simply passing through your membrane and not getting immobilized. Of course this would not be the case if your controls are also <200 bp, but they are larger then you may want to either change your membrane, or get controls that are of about the same size as your pre-miRNA.
My two cents.
Posted 05 November 2010 - 04:12 PM
Thanx for your inputs. My positive control is about the same size that of the target and I do run a miRNA ladder which is much smaller than the target pre-miRNA and I get the appropriate bands when I probe it with the same. whats more intriguing is that even if I use the same primers as a probe for the hybridization (37 degree C) which I use for RT PCR, I dont get a band.