Lamin A/C Positive Control siRNA - Has anyone used it? (May/03/2005 )
Was just wondering if anyone has used the Lamin A/C siRNA positive silencing control from Qiagen, what they thought of it and whether anyone could reccommend a good western blotting antibody for Lamin A/C? I am thinking about getting it and was just interested in other peoples opinion!
you can read this paper "Identification of essential genes in cultured mammalian cells using small interfering RNAs.
lamin A/C siRNA target region (NM_005572) 608-630.
The Qiagen oligos are the same sequence as on the Tronolab siRNA database so hopefully they will work, I was more interested in whether anyone had used the qiagen oligo as I have their negative control and I am not overly impressed, but that could be a bad workwoman blaming her tools! I am also trying out the Lamin A/C antibody from SantaCruz (clone 636 I think)...appears to be cleaner than others on the data sheet but we shall see.
Thanks again for the link
The Sanata Cruz antibody 636 was the one we settled on, but whether you saw LaminA, Lamin C, or a variable intensity of both seemed to be cell-line-dependent. In human carcinomas, detection of LaminA seemed the most reliable with this antibody.
If you're going to get your siRNA from Qiagen, the only thing I'd say is to pay attention to the fact that they offer two purity grades - 'HPP' and 'HPLC', of which HPLC is much purer. Both grades are available for custom siRNA, but a quick look at their website and it seems to me that if you order it as a 'validated control siRNA' you can only get the lower-purity HPP grade [quoted as '>90% pure']. We've had problems with the HPP grade [for custom siRNA], although this may be dependent on the cell types we work with. Hence we don't use it, as something about an siRNA that is only ~90% pure bothers us [what's the other 10%? Presumably premature termination products of the synthesis process].
If this makes you uneasy, you could try the LaminA/C control siRNA from MWG [Dharmacon if you're in the States?]. It's HPLC grade and we have always found this to be an excellent [if not cheap] control siRNA, but as I'm writing this at home and not work I can't verify the target sequence for you, you'll have to check that yourself.
Thanks for the advice! Whilst attempting to optimise the LaminA/C antibody and detecting no band it became clear that Jurkats don't express LaminA/C. Looking into it further I found that quite a large subset of cells don't express LaminA/C and only express B-type Lamins, including cells of the immune system, pancreatic islets and Purkinje cells so I am guessing I am going to have to find another positive control that is well expressed in T-cells. Any suggestions? I was thinking Vimentin which Amaxa use in their T-cells/T-cell lines but it bothers me that it is secreted by macrophages and I don't know if anyone has tested whether it is also secreted by T-cells. I was also thinking CD4 but I am not sure how well Jurkats express CD4, I think the levels are really low, I guess I will just have to run the through the FACS!