Can I use housekeeper like ß-actin as endogenous control for miRNA-qPCR ? - (Feb/18/2015 )

Hey everybody,

I would like to do some analyses on miRNA expression. I read a lot about normalization. I know you can use some snoRNA or spike in a synthetic RNA. But why is it not possible to use a “normal” housekeeper like ß-actin  or something like that? If you are analyzing tissues, where you housekeeper is expressed, why you cant use it as endogenous control?

I know that the “normal” housekeeper are difficult when the analysis is about blood, plasma or serum. But I don’t get the point when the analysis is in tissues.

 

Thanks for your help!

Best regards

You should use a stable miRNA for normalization of your miRNA.  Check out this paper. Also,  I believe 18s rRNA is more stable than actin or gapdh for normalizing mRNA RT-qPCR. 

Normalization of microRNA expression levels in quantitative RT-PCR assays: Identification of suitable reference RNA targets in normal and cancerous human solid tissues

Hey,

First of all thanks for your answer. Can you explain why I should use a miRNA for normalization in miRNA experiments? Why the normaliser has the be from the same class of RNA?

Of course I need a stable gene for normalisation. And if a miRNA is stable it is good to use it for normalisation in my miRNA experiment. But where is the difference between a stable miRNA and a stable “other” gene (no matter if ß-actin, gapda ,18s rRNA or whatever is stable in your tissuse). I just want to understand the difference in general. Or is there no difference?  .

 

I know that the “other” genes are much longer in their length. In the paper you can read “In addition, 5S rRNA (121 nt) and U6 snRNA (RNU6B, 45 nt) were included in some cases owing to their purported expression stability and use in several published miRNA qRT-PCR studies“. Beside the fact of the question of stability, 5s rRNA is with 121 nt quite long in comparison to miRNA. So the length is not that important?

 

May I just have a mistake in thinking, but I don’t get the point.

 

Thanks for your help again!

 

Best regards!

I do not think the selection is made on a single factor alone. However, I believe that selection of control that is similar size to miRNA is important because it confirms successful isolation. Depending on the isolation technique, a 20bp vs 1000bp - a 1000bp sequence could be isolated more efficiently than a 20bp sequence with a particular method. The most important thing is to use what you trust and use the control that is the most accepted in the field based off the literature. Are you planning to use LNA probes?

Hallo,

 

thanks again for your answer.

If I’m able to get a signal from my miRNA in qPCR, is that not enough to confirm a successful isolation?

My plan is to isolate total RNA that contains miRNA with TRIzol. MiRNA analyses should be done with a commercial kit (first polyadenylation of miRNAs, cDNA synthesis and detection with SYBR green).

Even if a 1000bp sequence is isolated more efficient than a small one, the proportion should be the same in all analysed samples. Or am I wrong?

 

I just want to get the point why nobody is using one of  the “other” genes for normalisation.

Some people are using TRIzol isolation in combination with columns. So these people only get small RNAs as the columns are made for this. In that case of course you can’t use a “normal” housekeeper and have to use a small RNA as control.

 

In my project I want to isolate total RNA including small RNAs from different tissue, so in theory I do have my “other” housekeeper for analyses. In contrast I also want to analyse miRNAs from plasma. There is no control and have to spike in something.

 

I personally don’t get the point why it is not popular to use a “normal” housekeeper if the analyse is made in tissue and total RNA is isolated.

I’m only searching for the right answer to understand that point. In my opinion there is in principle no difference. But may I’m totally wrong....

 

So thanks again for your help!

Best regards!

Have you seen this link? It might help you.