vacuum inflitration impact negatively on gen expression by changes the the three-dimensional (configuration) of transcriptional factors that responsible about the initiation of expression  

Thank you very much for your reply!

In my opinion its not so black and white. Vacuuming is a stress for any tissue including plant tissue and therefore it will affect gene expression, positively in the case of some genes (stress responsive genes for instance) and negatively for others. I dont think that overall gene expression should go down...Do you have evidence that it does ?

Hi, Hugo, I am also not very shure about this. I was doing uptake experiment with labelled sulphate and I use the method of vacuum infiltration of leaves to exchange the air from apoplast with a incubation solution. I was able to analyse Sulphate flux.

Then I used same material to analyse gene expression. I am struggling to get results from RT-qPCR. I isolated RNA got nice amount (from 200 up to 600ng /µl 260/280 and 260/230 ratio is also nice, I had two bends when I was checking RNA on the gel by electrophoresis. 

I used 1µg tóf RNA for synthesisi of cDNA. When I am running RT-qPCR I am getting very high Cp values (29-34) even for genes which showed high expression on the gel plus, the melting curve is very messy and I am getting multiple peaks. 

I was checking more things but always is the same. I am trying to figure out if my cDNA is a problem. and then, it came up on my mind what if this vacuum infiltration step can damage genes?

I am working with  Arabidopsis thaliana Col-0 and its mutant.

I could not found literature which will give me answer on this question but I found some literature where they are using Agrobacterium vacuum infiltration to introduce gene of interest and later analyse gene expression. But of that specific gene, I do not know what is happening with a indigenous and their expression and if their expression is somehow influenced negatively by infiltration step.

I am very confused and lost must confess.

All the best!

S.

The messy melting curves is problematic, try to dilute more your cDNA or use new primers (I would start by diluting and just run a gel rather than a costly qPCR to make sure you have a single band). cDNA can be diluted 10 to 100 folds to avoid PCR inhibiton froim RT mix. If you manage to get clean melting curves and still have all genes looking downregulated it could be that your reference gene is itself upregulated by vacuuming, making all other genes looking down regulated, then it would be simple just to use another ref gene.

Good luck.

Dear Hugo,

I do not know what is a problem. I did also normal PCR to check primer pairs of my genes and they looked fine on gel. I am sending here picture of them. Two of them look not so nice but others look fine.

 Do you think that even I am getting high Cp values (genes might be downregulated) of my genes of interested I should find  reference gene which showing also high Cp values and in that case I will be able to calculate gene expression? 

I am also thinking if I had sulphate uptake it means that genes responsible for sulphate uptake and assimilation were expressed.Otherwise, how would I explain than the incorporation of labelled sulphate into proteins, glutathione and glucosinolates...

Thank you for your help!

All the best!

S.

Your amplification looks good, single band (for most), I am surprised you have weird melting curve. The gene you amplify is it from a gene family ? Then a similar gene could give a band of same size but have a different melting curve...