qRT-PCR - HELP: diluting samples (Sep/14/2005 )

Hi,

I am new to the quantitation of gene expression via RT-PCR. I am a bit confused about why I have to dilute my RNA samples prior to RT. When one assume that 1 ug RNA is converted to 1 ug cDNA why can't I not simply make a dilution series of my cDNA and use that for the PCR step?

I want to perfom relative quantification using the comparative method. Thus, I have to do validation experiment first showing that the amplification efficiency of the target gene is approximately equal to the amplification efficiency of my reference gene (in vitro transcriped RNA). In order to do so I should look at how the delta Ct values vary with template dilution (RNA or cDNA dilution???)
I hope I got this right so far...

At what point do I have to dilute my samples and what should be the range of the dilution series?

Thanks for any help!!!!
Susan

one of the trickiest things to control in qPCR is RT efficiency; I dilute my RNA samples all to the same concentration prior to the RT reaction and then make a mastermix for all of the samples I will be assaying at the same time...then add the same small amount to each PCR replicate sample when setting those up

validating for delta-delta-Ct is a necessary evil, but once your'e done you can cruise through the rest

ABI's website is sometimes a PIA but if you can locate USER BULLETIN #2, follow the delta-delta-Ct validation format as close as you can for your system and your results should be good

at least that is what works for me

setting up the spreadsheets to do all my calculations for me was really the hardest part, but apparently there's some freeware called QGENE that is already set up (I can't remember the site, maybe Biotechniques?)

good luck

Hi aimikins,

Thanks for your response. I got the TB#2 but I am still not sure if I understand you right. Sorry.

So you dilute all your samples to an equal concentration and then use the same amount of RNA for each RT. Based on the assumption that 1 ug RNA is converted to 1 ug cDNA you set up your dilution of your reference and your target sample for the validation experiment. Right?

In the manual they speak of diluting RNA... so this is just so make it simple? HELP! Nobody in my lab has ever done qRT-PCR.

I got another question. My reference is an in vitro transcript piece of RNA that carries the same primer binding sites as my actual target (I will use gene specific primer). So my standard RNA sample is probably much higher concentrated than my actual target in the total RNA sample. Should I dilute the RNA prior to RT to let's say 10e-10?

Thanks again.
Susan

OK...I would not recommend diluting your RNA samples 10e-10

Do you quantify your samples?

This is what I do and it works quite well for me:

1. treat my cells
2. prep my RNA (stratagene Absolutely RNA miniprep kit; I do NOT use the DNAse step). I elute with a large volume and then precipitate and concentrate it down; through trial and error I have found that this gives me the largest yield
3. I add TE to dilute the RNA samples to 25ng/ul
4. I use 250ng (10ul) per RT/50ul total volume
5. Usually I put 5ul of the RT into 50ul/PCR reaction, which I split into two replicate samples. It's all about replicates with qPCR.

Serial 1:1.5 dilutions of template, as well as multiple dilutions of primers, were used initially to validate the delta-deltaCt and determine reaction efficiencies. The tricky part was finding amounts of template and primer that gave the same reaction efficiency for all 6 of my genes (for my system, I can get them all within 2.5% of each other but it took a lot of work in the beginning)
I use B actin as a reference gene.

This setup will not necessarily work with your given genes. You will have to determine your amounts empirically. To compare one sample with another, (and not just two genes in the same sample) you will have to do everything you can to make sure you are adding the same amount of template into each PCR every time you do it. That is why the specific careful dilution of the RNA at the beginning, and not just sort of randomly diluting it.


As far as your reference being more concentrated, what do you mean? your reference gene is more abundant than your target? the sample you are using as your control has more RNA in it? what exactly are you asking?
and how do your primers for both genes have the same sequence, but you will solve it with gene-specific primers? i am confused.

ok, hopefully I've been able to help

Hi,

Thanks again for taking the time to answer. I guess my last response was a bit confusing. As for my reference or standard (does it mean the same?) during PCR:
I PCR amplified a fragment of the target gene and fused a T7 promoter site to it in order to in vitro transcibe the DNA fragment into anitsense RNA. I purified the RNA transcript and determined its concentration. The in vitro RNA transcipt is targeted by the same pimers I will use for my actual target mRNA. With "higher concentrated" I do mean it's more abundant in the reaction because it is just the pure transcript in the tube and not total RNA.

I thought I have to do the following:
1) After isolating and quantifying total RNA and in vitro transcribed RNA I set up 2 RT + RT controls (without enzyme and without RNA).
I use the same amount of RNA (carefully diluted to the same concentration) for both my control (in vitro transcript) and total RNA sample (target).

2) I prepare a dilution series of both the control cDNA and the target cDNA
3) I run the PCR
4) I pray and hope I did everything right.

I guess I am not totally clear about what I am going to do :-(.

Susan

Susan -

it looks as though you are doing it right. are you having problems? or do you just want to make sure? have you optimized your primer concentrations as well?

how are your runs looking?

Aimee

i haven't done anything so far. I just wanted to make sure before I move on to the practical part. Thank you so much! It helped me a lot to have someone to talk to about this. It's a bit frustrating if nobody else in the lab has an idea what you are doing.

Susan

Susan - I hear you.

I was in the same boat about 20 months ago

the learning curve really sucked but now it goes like hotcakes...quick and reproducible every time

don't forget to optimize your primers, that step is huge and just as important as amount of starting template in determining reaction efficiency. and it has to be done for real-time; what works well for straight PCR may not give you what you want when you toss it in the old 7000

good luck

It is nice that Susan has solved her problem. here I have a site where you can download software for calculation: http://rest.gene-quantification.info/
For honest, I have not try it myself because my job they have already created one for their. But the software in this site are usful. Good luck!

Ribosoul

I have been using the REST method for a while now and although it was a bit confusing in the beginning (when I downloaded the excel file template... you have to be careful to make sure that you are inputting the correct data in each column) but once figured out it makes life much easier. I definately recommend it and so would all the other people in my lab who have been doing qPCR for a while now.

Good luck. I find the primer QC steps are the worse!

Auriol