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using cell line to generate standard curve

cell line standard curve real-time pcr

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6 replies to this topic

#1 detriar

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Posted 14 April 2013 - 04:46 AM

Hello all,

I'm a beginner in real-time pcr. I wanna ask a question: if I'm going to do an absolute quantification to measure copy numbers--let's say--of gene X, could I use cell line instead of plasmid to generate standard curve for this research? I'm planning to do RNA isolation from frozen tissues, then making the cDNA, and that cDNA will be the unknown sample.
I've been searching articles or journals related to this problem (cell line for standard curve), but not yet have an answer, or maybe I looked at the wrong place. Could anyone suggest articles or journals related to this?

Thank you so much all.

Cheers,
Dee

Edited by detriar, 14 April 2013 - 05:58 AM.


#2 bigudukaz

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Posted 14 April 2013 - 11:53 PM

The main thing is, that you have to know exact quantity of copies in your standard curve. Dont use plasmid as it will not have to undergo the cDNA synthesis step. Find the cell line, that expresses the gene and use it for standard curve. You will have to know the expression levels of that gene so you can calculate the number of RNAs of your specific gene.
Treat your samples the same way as your standards.
Pretty much any cell line that expresses that gene in known quantities should be good for standard curve.
You will have to quantify the extracted RNA and, i think in your situation, use an internal control to be able to see the difference in amounts of cells in samples this way you wont need the standard curve.

To get around the standard curve in other way is to use digital PCR (from Biorad i think), but it will get expensive if you dont have the equipment.

#3 detriar

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Posted 15 April 2013 - 03:50 AM

bigudukaz,bigudukaz

thanks for the insight, I really appreciate your response Posted Image

Now I'm a little confused here, whether I use this formula:

(X g/μl RNA / [transcript length in nucleotides x 340]) x 6.022 x 1023 = Y molecules/μl

or this formula:

(X g/μl DNA / [plasmid length in basepairs or length of PCR products x 660]) x 6.022 x 1023 = Y molecules/μl

to generate the standard copy numbers from each its cDNA concentration, If I want the final result of the research in copy numbers RNA/ul?

Thank you.

Edited by detriar, 15 April 2013 - 03:54 AM.


#4 bigudukaz

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Posted 16 April 2013 - 01:24 AM

when you extract the RNA - you get all of it, there are special spin columns for tRNA and others. The problem is - you have to know how many RNAs is of your interest. There is pretty much only one option that is normaly used - to compare interest gene to the control gene (the gene that is expressed in same levels in all the cells - samples and controls) so you can relatively compare expression over the internal control gene expression (the control could be GAPDH or you can look for others as different cell lines might express it differently).

As i can see you want to go directly to the numbers. The problem is - the cell might have a lot of RNAs of your interest so small increase of cells will give big impact on specific RNA count. So unless you can make sure, that you take same amount of cells that are in the same stage of growth and undergo the same procedure with the same losses and other "same" things :)

As for your choice of calculations - it depends of what you are using for std curve:
Plasmid - go with the length of plasmid with double helix, so 660,
PCR fragment - PCR fragment length and its double,
If you go with RNA - then its 330 as it is single strand.
Basically - count how long the "thing" that your fragment is in and whether its double or single strand.

Strange thing that you are going for number of specific RNAs in the sample... it will be good only for positive or negative expression, but not for quantification...

If you have more questions, dont hesitate to ask.

#5 detriar

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Posted 16 April 2013 - 03:53 AM

bigudukaz
once again thanks for the response,
I'm sorry I think I didn't make myself clear before..
If I isolated the RNA of the sample --> then I synthesize the cDNA --> real-time PCR step,
so I have to do the same path with the standard (cell line), right?
I have to isolate the RNA of the cell line --> then I synthesize the cell line's cDNA --> measure its concentration by spectrophotometry --> the result (ng/ul) will be used to find out its copy numbers/ul (with Avogadro) --> making serial dilutions of the cell line --> the log copy numbers will become the x-axis of the standard curve.
(And then come the normalisation steps....)
Are those steps above right?

And about why I intend to know the RNA copy numbers of the sample, yes you're right, I want to know the number of specific RNAs in the sample,
because the principle investigator of this research wants to know the difference of RNA copy numbers of sample before and after some treatme nts.
That's why I'm going to do the absolute quantification real-time PCR.

I feel challenged with this research but because I've never done it before I think I have to ask somewhere Posted Image , so that I'm sure that the methods I'm going to do is clear.
Moreover the absolute quantification depends on the 'goodness' of the standard curve, so this step is very crucial I think.

Thank you, again Posted Image

#6 bigudukaz

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Posted 16 April 2013 - 11:17 PM

There is a problem on the sample preparation as you need to "loose" the same number of specific RNA coppies.

Probably the best way would be to know what % of RNA is your specific RNA in standard cells so you can get ng/ul ->coppies/ul. If you count it like this straigth (after extraction) from concentration - you will not get coppy number of your RNA as nanodrop shows you TOTAL RNA.

Lets say you get cell line that expresses gene X at 10^6 coppies per 1 ng of total RNA. From there its easy. the problem is to get those cells, preferably that cells were as simmilar to your cells as possible (proteins can interfere with extraction).

I suppose you will be using spin columns for RNA extraction? :D As the good "old" methods are not that good for qPCR (well at least basing on my experience).

#7 detriar

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Posted 23 May 2013 - 08:39 PM

Many thanks bigudukaz





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