Detection of LNA miRNA knock-down by qPCR? - (Feb/01/2008 )

Hi All,
I have been trying to knock-down miRNA in cell line (high endogenous expression) using LNA (5'-FAM labeled) and over-express in other cell line (low endogenous expression) along with relevent controls (scrambleMIRS). Problem is whilst I can see over-expression very clearly by qRT-PCR (Taqman), the knock-down is less convincing. There are two possible senarios to my mind 1) that qPCR doesn't work to measure knock-down or 2) that I'm not getting sufficient knock-down to measure properly.
I have looked in the literature and can only find knock-down validated by Northern, I don't wnat to do this as I don't have enough material. I think the LNA-miRNA hybrid should be stable enough in the qPCR reaction so that the miRNA is no longer available for binding to loop primer so should not be detected and so knock-down measurement should be possible by this technique. Any ideas?

On a related point does anyone know what happens to the miRNA during knock-down is it degraded as double stranded or does it hang about the cell non-functionally?

I also have tried to use 5'FAM labelled LNA/mimics to measure tranfection efficiency but fluorescence is not strong enough to tell above background and can't be used for cell sorting (am using suspension culture)- anyone have any ideas how to measure efficiency?

Many thanks in advance.
pBad

ii was my understanding that qPCR is more accurate than Northern. so most likely explanation is that you dont have a sufficient knock-down. Maybe there is an issue with getting the LNA probes inside the cells? are you taking in account natural abundance of miRNA --- that would be important when deciding the concentration of the probe to transfect. Can you measure LNA probes by qPCR? that would give you a copy number per cell.

good luck

Northern gives you size information about the nucleic acid target, while PRC gives a defined PCR product size. If you are blocking maturation of a miRNA with a steric-blocking oligo, you likely have pri-miRNA accumulating. This can give a PCR product even if there is no mature miRNA present. A Northern allows you to differentiate the mature miRNA from the pri-miRNA.

For more discussion, see:
http://biology.plosjournals.org/perlserv/?...al.pbio.0050203
Kloosterman WP, Lagendijk AK, Ketting RF, Moulton JD, Plasterk RH. Targeted Inhibition of miRNA Maturation with Morpholinos Reveals a Role for miR-375 in Pancreatic Islet Development. PLoS Biol. 2007 Jul 24;5(8):e203 [Epub ahead of print]

Do you see any phenotype with the knockdown? Because if you do, but you don't see knockdown by qPCR then my best guess would be that the miRNA:LNA hybrid stays in the cell for some time (i.e. the miRNA is not degraded, just sequestered (i.e. think of it as a miRNA sponge)). Even though the LNA has a high affinity for the miRNA, wouldn't the high temp denaturing step of PCR (and possibly the salt conc) result in it's dissociation, so that it is detectable by qPCR?

The TaqMan PCR is specific to mature miRNA, so you are not detecting any accumulation of precursor molecules.

Edit to add: I just thought, that the hairpin primer binds the mature miRNA during the RT step, in which case I guess the low temp could (or would?) mean that the LNA is still bound to the miRNA. In this case you should be able to see a knockdown if it worked (theoretically!)

Hi everyone,

I'm also having a problem detecting miRNA knockdown by LNA, using qPCR, but can detect overexpression of the miRNA. Has anyone been able to succesfully detect knockdown using qPCR- SYBR or Taqman? Another strange thing is that my GAPDH qPCR control seem to be affected by the transfection with both the miR-LNA and scramble LNA; could this be a non-specific effect, and has anyone had these problems before? (GAPDH is not normally affected by other transfections...)

Many thanks in advance...
madpie

Here are my two cents. Please correct me if I am wrong.

1) LNA-miRNA binding may not lead to degradation of the miRNA. Therefore it makes sense that you could not detect any change by qRT-PCR.

2) The reason Northern "detects" that is because the Northern probe could no longer bind to the RNA-LNA duplex, which is not disrupted even in the denaturing gel. (Naguibneva et al, Nat Cell Biol 2006)

Hi everyone,

I'm also having a problem detecting miRNA knockdown by LNA, using qPCR, but can detect overexpression of the miRNA. Has anyone been able to succesfully detect knockdown using qPCR- SYBR or Taqman? Another strange thing is that my GAPDH qPCR control seem to be affected by the transfection with both the miR-LNA and scramble LNA; could this be a non-specific effect, and has anyone had these problems before? (GAPDH is not normally affected by other transfections...)

Many thanks in advance...
madpie