WHAT and WHY Normalization?

[HTS76]

What is normalization of data and why do we care for it?

[new2science]

Hi,

I'm new to this site and I hope this is where I should be posting? I have a question about normalization. I was told to normalize my samples to 1ug/ul but I do not know how to do this. I just purified plasmids using a QIAGEN maxi prep kit and now I first have to measure the concentration of each sample I have but then I was told to normalize my samples to 1ug/ul. How do I do that? I don't know how to normalize my samples. Thanks.

[danfive]

Originally Posted by new2science

Hi,

I'm new to this site and I hope this is where I should be posting? I have a question about normalization. I was told to normalize my samples to 1ug/ul but I do not know how to do this. I just purified plasmids using a QIAGEN maxi prep kit and now I first have to measure the concentration of each sample I have but then I was told to normalize my samples to 1ug/ul. How do I do that? I don't know how to normalize my samples. Thanks.

OK, normalize, may not be the best term in your case, but they mean dilute your different samples to the same concentration of 1ug/ul.

so if sample 1 comes out to be 50ug/ul dilute 1:50 and get 1ug/ul
if sample 2 is 25ug/ul dilute 1:25 and get 1ug/ul
and so on.

[muffinman563]

Originally Posted by HTS76

What is normalization of data and why do we care for it?

If you want to understand why normalization is done for PCR assays, have a look at
'Real-Time PCR
Applications Guide' from Bio-Rad, i have this PDF but don't know its html...sorry but you will need to snoop around this, start at the biorad website. I can send it to you if you give me your email. Basically it controls for experimental variance i.e efficiency of cDNA extraction vary in different samples ( say cancer patient 1,2 etc )of same group ( say cancer) as well as controlling (normalizing better term) for biological variance ( maybe patient 1 >expression of PCR target slightly more relative to patient 2)

Usually these genes you normalize to are similar in expression across the groups ( i.e cancer vs non-cancer) , GAPDH and B-actin are some common ones referred to as reference genes.

Also useful-


"Critical Factors for Successful Real-Time PCR - English (PDF)"

QiAgen websites : Literature> Brochures & Application Guide

Sorry can't post the link as im <5 post

[SebaQ]

Well, I've been told a few things about normalization, and this I can share:

Normalization of data means to "standarize" the data to a fixed parameter we know. I guess it's better with an example.

If you're trying to check whether certain stimuli affect the transcription of a certain gene, you can check it doing a RT-PCR or a Northern Blot. When you do this experiments, you are told to do it for your gene of interest and for a housekeeping gene (normalizing gene).

Let's say you performed your PCR and worked out great.

You check the amplification of you gene of interest in the sample obtained from the stimuled organism and compare it to the same gene in the non-stimuled organism, don't you? Fine. Now you have to compare the bands for the housekeeping gene. There are two possible scenarios for this:


- The housekeeping gene bands are the same in concentration when analized in the gel and the gene of interest bands are automatically comparable

- The housekeeping gene bands are not the same in concentration when analized in the gel, and therefore you can't just compare the bands of you gene of interest.

We use a housekeeping gene we know it won't be affected by the stimuli we are studying, because we need and expression and loading control. Expression, because we need to know there is the same quantity of total RNA (cDNA) analyzed kwnowing it won't change its expression due to the stimuli, and loading control, because you could also state this difference in the analyzed are due to gel loading problems . We must have a tool that allow us to surpass all these possible hypothesis of why we are seeing differences in the gene expression and state that the difference observed is ONLY due to the stimuli applied.

You have to normalize the housekeeping gene because you do know its expression isn't affected by the stimuli you are studying. That's why you normalize. To adjust the parameters so you can analyze directly what's of interest for you.

Mathematically, if you have these results [I'll just put numbers without units]

Stimulated organism:

- Gene of Interest: 3
- Housekeeping gene: 1

Non-stimulated organism:

- Gene of interest: 1
- Housekeeping gene: 1

You can say the stimuli caused a threefold rise in the transcription of you studied gene.


But if you have

Stimulated organism:

- Gene of Interest: 3
- Housekeeping gene: 3

Non-stimulated organism:

- Gene of interest: 1
- Housekeeping gene: 1

The ratios 3:3 and 1:1 are the same, so you can't say anything about this result, because no one could say if the difference seen is due to higher cDNA concentration when the PCR was performed, or you just loaded three times volume in the agarose gel.

Well, sorry for the extension ^^! Hope it'll be useful

Best Regards!