Real time PCR - RT-PCR vs Western blot (Dec/26/2007 )
Hi Every one,
I am new to R(eal)T-PCR,
When I did western blot (at protein level) I saw significant increase of one of my X protein in treatment group than control group. But its not showing any change at mRNA level ( when I did Real time PCR). what could be the reason? any Idea? Please help me
they are not measuring the same thing. usually there is a correlation, but it's not a guarantee.
there could be differences in post-translational modifcation and processing that allow for the differences in protein levels
first, however, how are your real-time results? efficiency? melting curve? reproducibility? controls behaving as expected?
Thank you for your reply
Melt curve was good, no primer dimers were there. in terms of reproducibility... i am seeing inconsistancy in results. I am using beta actin as a house keeping gene. It was good but the problem is,I am seeing unexpected results in control group.
I am thinking of doing following things.
I will calibrate the machine since I moved my Biorad machine. may be it is not catching right signal.
I would like to check with positive control. and see if i get expected results.
I have primers which have already used in insitu hybridization. I am not sure if I can use them for real time pcr?
Please suggest me if this is a right approach
Have you check mRNA stability?! or protein turnover?! Can you use an inhibitor of trancription to see if that increase protein levels are due to transcription?!
About the primers... maybe its better to design new primer for RT-PCR... althoug... differences should not be primer-dependent.. but if the primers that you are been using are not proper... you have to be sure about that!!
Thank you for the reply
I am not sure how to check protein turnover. Can you please let me know how to check protein turnover?
I made cDNA after I check m RNA quality in nanodrop. I saw 260/280 was below 1.8 (it was between 0.69 -1.15). I have not purified RNA and I have not used any columns are methods which help to purify RNA. I am little worried if I use RNA purification methods I will loose RNA. I extract RNA from tissue using Trizol. I am getting very little amount of RNA. I made cDNA right after I normalize RNA.
Does low 260/230 ratio effects my PCR results?
yes especially if your ratios are all over the place. a ratio that low suggests contamination; I would recommend doing what you can to increase yield and using a column to purify (Fred_33 probably can advise you best here; he has extensive knowledge of the Trizol method)
I would tell you that if you get a batch of RNA at a ratio of 1.5, and another batch at 1.8...your relative amounts of contamination are different. therefore you will not be adding the same amount of RNA to your amplification reactions. therefore, even if everything else is perfect, you will still get inconsistent results. also, please ALWAYS include a positive control - if you don't have pos and neg controls, it's much harder to trust your results in an assay with so many, many variables.
I would not change your primers, unless you are getting poor amplification efficiency. I would work on improving your RNA quality and the rest should take care of itself.
We also use the Trizol to extract RNA. This method is pretty easy and low cost, but DNA contamination may be a problem. To solve this, we designed our primers on two intron/exon jonctions, to ensure that we only amplify the RNA.
And as said Aimkins, try to always include controls to validate your experiments. You'll save a lot of time
After I read your comments, I felt that my knowledge is very limited in RT-PCR. I am glad that I found this forum.
Recently I have purified my RNA and 260/280 ratio was >2 in control and treatment groups. I saw nice beta actin (house keeping gene) with out variations. But When I used other primers I saw primer dimers.
My Primers working concentration was 100nm for SYBR green protocol, and I used 58 as an annealing temperature. I was using 1.5% agarose gel for gel running. Product size was 113 base pairs
Any thing I can do it to improve? can you please see the image and suggest
We have noticed and hypothecized that when there are not a lot of target RNA, primer would tend more to form dimers. The best solution to primer dimer, to our knowledge, is still to design a new pair, since it is easy, rapid and costs about nothing.
And you did not join any image