
shRNA problem - Higher expression at the mRNA level !
#1
Posted 17 September 2012 - 01:05 AM
I'm currently trying to knock-down the expression of the FCGRT gene (encoding the neonatal FcRn recpetor for IgG) in liver cells, so I chose to tansfect them with some different shRNA-encoding plasmids provided by a supplier.
Since there is no efficient antibody marketed, I can't possibly check the diminution of the receptor expression, so I tried to check it at the mRNA level by RT-qPCR.
The problem is that, compared to wild-type cells, the mRNA level of FCGRT transcripts is higher in cells transfected with the shRNA control. Is it possible that the introduction of the irrelevant shRNA in those liver cell induced an upregulation of the gene ?
However, I managed to obtain some cells that seem to be knocked-down for the FcRn (still at the mRNA level), compared to shRNA control-cells.
My question is, do you think it is relevant to continue and try functionnal experiments on those FcRn-KO-cells compared to the shRNA control-cells, since the level of transcripts supposed to encode the FcRn receptor is higher in it than in wild-type cells ?
Thanks a lot !
#2
Posted 17 September 2012 - 08:06 PM
#3
Posted 18 September 2012 - 01:53 AM
I don't believe the problem is due to what you pointed out. In fact, the transfection efficiency was controlled (and cells were selected by their ability to resist under a certain dose of Puromycine, which resistance is given by the shRNA plasmids) as well the plasmid preparations. The PCR system, I do agree is not the best way to control wether there is an expression or not, but I can't do a WB since I have no efficient antibody against FcRn.
Moreover, the vendor ensure at least a 70% mRNA knock-down. So I manage to have some good FcRn-KO-cells compared to control cells tranfected with control shRNA, but the real problem is still that those control cells seem to express the FcRn receptor more than wild-type cells, and I don't know what it is due to ?
#4
Posted 26 September 2012 - 01:09 PM
#5
Posted 18 October 2012 - 11:52 AM
#6
Posted 09 April 2014 - 03:26 PM
This phenomenon can now probably be explained by the RNAa mechanism. In C. elegans, 22G-RNAs (which are secondary RNAs derived from piRNAs) antisense to endogenous mRNA are loaded by the CSR-1 Argonute protein to form a complex which then enters the nucleus. the 22G-RNA/CSR-1 complex binds to nascent mRNA to promote the transcription of mRNA epigenetically. You can read the following papers:
Seth M, Shirayama M, Gu W, Ishidate T, Conte D Jr, Mello CC.
Global effects of the CSR-1 RNA interference pathway on the transcriptional landscape.
Cecere G, Hoersch S, O'Keeffe S, Sachidanandam R, Grishok A.
Also tagged with one or more of these keywords: shRNA, RNAi
Protocols and Techniques Forums →
siRNA, microRNA and RNAi →
Where can I find 100-200 unique primer sequences for preparation of arrayStarted by Bio21, 16 Jan 2017 ![]() |
|
![]() |
||
Protocols and Techniques Forums →
Molecular Biology →
siRNA with expensive gentamicin-containing mediumStarted by JDSBlueDevl, 24 Mar 2016 ![]() |
|
![]() |
||
Protocols and Techniques Forums →
siRNA, microRNA and RNAi →
commercial siRNA causing off target effectsStarted by philman, 16 Sep 2015 ![]() |
|
![]() |
||
Protocols and Techniques Forums →
siRNA, microRNA and RNAi →
How to clone two mir-30 based shRNAs into the same vector and express them fromStarted by 5280, 01 Aug 2015 ![]() |
|
![]() |
||
Protocols and Techniques Forums →
siRNA, microRNA and RNAi →
Commercially stable shRNA cell linesStarted by BioPe, 12 Feb 2015 ![]() |
|
![]() |