3 replies to this topic

[Eon's mommy]

Hello everyone,

I have one question about making standards and their dilutions for Real-Time PCR. To be clear I will shortly describe my experiment.
Test subject: trout (A, B and hybrids AxB); in every trout group there are three subjects (A1, A2, A3; B1, B2, B3 and so on)
Tissue sample: skin
Objective: to confirm differently expressed gene between subjects

If I understand correctly you can prepare standards from basis samples, so-called calibrator or from independent sample as long it expresses both the target and endogenous control. I wil use calibrator for my standards (let us say that calibrator will be group A). And now finally the question   for standards it's better to use mixture of samples is that mean that I need to pool equal amount from each A sample (3) and make dilutions?
Would be very wrong if I use equal amount from every sample (9)? If I do that, then I can't say that standards are made from calibrator?
Please help me  

For the end just one little question about ideal dilution of samples for real-time PCR. Lab buddy told me that I need to dilute my samples so that the endogenous control Ct is 15-20 and sample Ct is 27-35. He is working with mice. Is there any written or unwritten rule about this?

Thank you for any info.

[umam]

I do a 1:1000 dilution for my samples (I work wit mosquitoes) and have a range from 1pg to 0.001 pg, ideally you should get a CT value of about 3  cycles per dilution. Eg: 1pg : Ct=16, 0.1pg Ct=19 and so on.
I hope this helps.

[kedar]

Hi,

I guess if you are checking expression of many genes, it's quicker to use relative quantitation method. If you are doing that, you don't need standards and all that!
I am not sure whether you are doing relative or absolute quantitation. If relative, there is no question of standards.
If absolute, there are no calibrators, just endo control which should be expressed in same amount in all your samples...there is no need to mix and all that.

'' Lab buddy told me that I need to dilute my samples so that the endogenous control Ct is 15-20 and sample Ct is 27-35. He is working with mice. Is there any written or unwritten rule about this?''-----
I think this is not relevant. I use mice and use HPRT gene as endo control. I don't care where the Ct is. I just care if the HPRT expression is able to normalise my target. that's all.


Kedar

Eon's mommy, on May 28 2009, 12:07 AM, said:

Hello everyone,

I have one question about making standards and their dilutions for Real-Time PCR. To be clear I will shortly describe my experiment.
Test subject: trout (A, B and hybrids AxB); in every trout group there are three subjects (A1, A2, A3; B1, B2, B3 and so on)
Tissue sample: skin
Objective: to confirm differently expressed gene between subjects

If I understand correctly you can prepare standards from basis samples, so-called calibrator or from independent sample as long it expresses both the target and endogenous control. I wil use calibrator for my standards (let us say that calibrator will be group A). And now finally the question   for standards it's better to use mixture of samples is that mean that I need to pool equal amount from each A sample (3) and make dilutions?
Would be very wrong if I use equal amount from every sample (9)? If I do that, then I can't say that standards are made from calibrator?
Please help me  

For the end just one little question about ideal dilution of samples for real-time PCR. Lab buddy told me that I need to dilute my samples so that the endogenous control Ct is 15-20 and sample Ct is 27-35. He is working with mice. Is there any written or unwritten rule about this?

Thank you for any info.

[Eon's mommy]

Thank you umam and kedar for your replies.

If I understand Real-Time PCR correctly we have two quantification strategies; absolute and relative quantification. Absolute quantification relates on calibration curve and standards, while relative quantification is based on the expression levels of a target gene versus reference gene (housekeeping gene, control gene). Relative quantification does not require standards. Ok to here is everything crystal clear  !

If you go through "User Bulletin #2" from Applied Biosystems, you'll see that you have two relative quantification methods; one is relative standard curve method and the other is comparative Ct method (ddCt).

Relative STANDARD method:
as it already says in the name you need some kind of standards to do this method. To be precise you need standard, calibrator and tester. There is no need for standard to be experimental sample, as long it expresses both the target and endogenous control genes. Calibrator is some basis sample (usully control) and your target quantities are expressed as an n-fold difference relative to the calibrator. You can use calibrator as standard as well. Tester is your target, treated sample.
For this method standards are required, but all you need to know is their relative dilutions.

Adventages: minimum amount of validation;
                   highly accurate quantitative results;
Disadventages: standard curves on each plate;
                       requires a lot of reagents;
                       if you are using calibrator as standard, you'll need a lot of calibrator (BIG problem if your
                       calibrator is in short supply).  

Comparative Ct method (ddCt method):
you can make it in two ways; with or without efficiency correction. If you are doing ddCt without efficiency correction you don't need standards. It is strongly recomended that you do efficiency correction because result are more reliable. For PCR efficiency you need to do, so called, validation experiment. In validation experiment efficiencies of amplicons are achieved by running standard curves. If the PCR efficiencies between the target and reference gene are relatively equal (absolute value of the slope of Ct vs. log input is < 0.1), only then you don't need to use standard curves.

Advantages: elimination of standard curves (as long as validation experiment is performed);
                   reduced ragent usage;
Disadvantages: inital validation

Ok this was short summary of "User Bulletin #2" by Applied Biosystems.  

And here comes my dilemma.
I will probably use comparative Ct method but I don't know which sample to use for validation experiment. As I said I have three groups of samples (A, B and C) and in each group there are three experimental replicates.
If I choose group A for my control and do the validation experiment, can I put equal amount of A1, A2 and A3 together, do dilutions and run Real-Time PCR? Or do I use just one sample (A1) and run Real-Time PCR?

Please   Real-Time PCR experts help me out! I am novice in Real-Time PCR and I would really like to know how to do it before I go to lab and actually do it   .