Smeary bands in real-time RT-PCR - (Jan/18/2006 )

Hi,

I am just starting with real time RT-PCR.
The first two assays that I have done, I found bands about the right size as i would have expected, but with a smear in the upwards direction (ie towards a larger sized product than expected).

Also, there was a faint smeary band in the RT control sample (though not in the no template control).
Does anyone have an idea what might cause this and also how much of a problem it is?

thanks

first, I would guess you have genomic DNA contamination of your RNA prep

what size is the faint smeary band with your no-RT control? the same as the smear in your 'positive' samples?

and, yeah, this is a problem because it likely skews your results upward (i.e. you think there is more transcript/mRNA than is really present)

do you run dissociation curves? what do you see, if you do?

first, I would guess you have genomic DNA contamination of your RNA prep

what size is the faint smeary band with your no-RT control? the same as the smear in your 'positive' samples?

and, yeah, this is a problem because it likely skews your results upward (i.e. you think there is more transcript/mRNA than is really present)

do you run dissociation curves? what do you see, if you do?

Dear Amikins,

Thanks for the reply.

yes, the faint smeary band in RT control is about the same size as the other smears.

Before I started, I ran the RNA out on a gel and checked it on the spectro. It had two bands (18s and 28s) exactly where they should be and no genomic DNA band. Could it still be contaminated?

I am just starting. What are dissociation curves?

thanks

first, I would guess you have genomic DNA contamination of your RNA prep

what size is the faint smeary band with your no-RT control? the same as the smear in your 'positive' samples?

and, yeah, this is a problem because it likely skews your results upward (i.e. you think there is more transcript/mRNA than is really present)

do you run dissociation curves? what do you see, if you do?

Hi again,

I also have another issue. I just checked the delta Rn curves.
In those that look as though they have worked, instead of reaching a plateau and staying there, they actually go down towards the end of the reaction. What could cause that?

thanks!!

I suspect you might need to back up a bit. is there someone else in your lab who works on real-time? and, are you attempting to quantify your transcript?

I would very highly recommend going to ABI (Applied Biosystems) website and downloading User Bulletin #2. If you do a search from their main page it will come up as a pdf. I cannot get the link to establish properly for you; sorry! But I think it would be good for you to read this bulletin; it answers many questions you may have about starting out and what you need to do to get trustworthy data.

I'm also a beginner, and I would suggest "The Guide for Performing Relative Quantitation of Gene Expression Using Real-Time Quantitative PCR" in addition to User Bulletin #2.

you can go here: https://www2.appliedbiosystems.com/support/apptech/#rt_pcr

Click on: Real-Time PCR / TaqMan® Genomic Assays, and it's the "Performing relative quantitation..." under Applications.

Thanks,

I will read those. No, there is no one else performing real time PCR here. I am the only person in my lab doing molecular biology, the others do enzyme assays and physiology. It is tricky as I am more or less teaching myself this stuff.

Hence this forum is really useful!

I have read various guides but it doesn't seem to stick with me until I am actually trying it out, and then I understand.

Eventually I would like to quantify.
At the moment I am just trying to get a consistently amplified product on this real time machine. I know my particular protocol (including primers etc) has worked previously on another real time machine for a former employee of my boss. Now I have taken over her job and need to get it running on the new machine before I can do anything else.

thanks again

Hi again,

I also have another issue. I just checked the delta Rn curves.
In those that look as though they have worked, instead of reaching a plateau and staying there, they actually go down towards the end of the reaction. What could cause that?

thanks!!