Enrichement on specific RNA from sinaptoneurosomes - Problem

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Ana Rita
Ana Rita's picture
Enrichement on specific RNA from sinaptoneurosomes - Problem

ello,

I am isolating synaptoneurosomes and the next step is to check for the quantity of some target RNA; however, 1st i need to show that synapton have more relative quantity of some RNA when compared with the homogenated. For instance, mRNA for PSD95 should be more present when compared with the homogenated preparation.

But, is happening the opposite. The difference is not that much, but still i have more expression of PSD95 in homogenated than in synaptoneurosomes. I have done tha same for protein and did go very well.

I am just guessing that the proportion of RNA in such a small structure when compared with the whole cell is obscuring my results.

Does anyone have a clue?

Ivan Delgado
Ivan Delgado's picture
 

 
Hi Ana Rita,

When you say that you are getting more PSD95 expression in homogenate as opposed to synaptoneurosomes, the first thought that comes to mind is: how are you measuring expression? While I assume you are adding equal amounts of RNA from both samples, the fact is that you need to normalize the expression of PSD95 within each qPCR experiment in order to be able to compare homogenate against synaptoneurosome. In other words, you need to run both a PSD95 assay and a reference gene assay for each sample. Standard reference gene assays include 18S, GAPDH, beta-actin, etc. Once you run both assays for each sample, then you normalize the PSD95 signal to the reference gene assay signal for each sample. Only then can you compare homogenate versus synaptoneurosomes. 

Hope this makes sense.

Also, have you measure the efficiency of your PSD95 qPCR assay in each sample? If your RNA samples are not the same (equal level of purity), then the efficiency of the assay will suffer and you can get very different results for each sample even thought in reality they contain the same level of PSD95. To determine the efficiency of a qPCR assay all you need to do is run about 5 dilutions of your sample and analyze the results using a liner regression. Most qPCR software can do this automatically.