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Steps in performing qRT-PCR for microarray validation

microarray validation standard curve in qPCR relative quantification qRT-PCR steps in qPCR

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#1 Neanderthal

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Posted 06 March 2013 - 07:46 AM

Dear All,

I am going to do qRT-PCR for the first time to validate the microarray data, so I need all your help in designing and executing the steps involved. I highly appreciate all your responses and am looking forward to it!!

Here is some information:

1. I've two cell lines (C1 and C2) both having control and treated (2 treatments each) samples (C1C, C1T1, C1T2, C2C, C2T1, C2T2). Both treatments have the same control for one cell line (i.e C1C is the control for both CiT1 and C1T2; and C2C is the control for both C2T1 and C2T2). I am looking at the gene expression changes between treated and control groups (i.e C1C vs C1T1 and C1C vs C1T2, etc.) in both cell lines (so that I can compare with the microarray data that I already have).

2. I am going to use two-step RT PCR protocol and SYBR-Green chemistry ( using Power SYBR Green PCR master mix from ABI # 4367659).

3. I've around 5 genes (my targets of interest) per treatment (excluding house-keeping gene, which by the way, will be GAPDH), so a total of 30 genes (targets) to be validated. I've 100 pmol/uM stocks of primers for these genes.

4. I want to use relative quantification (either standard curve or delta-delta CT method) for data analysis.


So, based on these information ( please ask me if anything more is required), can somebody please explain to me the steps to be done for this experiment. Some specific questions that I have are:

1. What should be the first step - primer optimization or the target cDNA optimization (for assessing dynamic range, assay precision and efficiency)- and what are the steps involved?

2. If I want to use relative quantification using standard curve, what should be my calibrator? If I am right, I think in my case, I have to use 2 calibrators since I have two cell lines and hence my control(i.e un-treated) samples will be my calibrators. i.e C1C will be the calibrator for both C1T1 and C1T2; and C2C will be the calibrator for C2T1 and C2T2. Is that right?

3. If I want to use relative quantification using standard curve, which cDNA should I use for making dilutions for generating standard curve? Can I use dilutions of C1C for generating standard curve (for both C1T1 and C1T2) and dilutions of C2C for generating standard curves for both C2T1 and C2T2? I've seen people pooling equal amount of cDNA from all their samples and then diluting it to make standard curve. Should I pool my samples in that case? (in which case, will it make sense if I pool C1T1 and C2T2 for generating standard curve for cell line C1 and C2T1 and C2T2 for cell line C2?). I don't think this makes sense because I want to compare C1C vs C1T1 and C1C vs C1T2 so may be I should pool C1C and C1T1 as one (std for treatment T1) and then C1C and C1T2 as another(std for treatment T2). Is this ok?

4. Also, if I want to use relative quantification using standard curve, can I use the same standard curve for all my targets (~ 20 genes) if I run them in a single plate? If the targets are continued in a second plate, should I again put these standard dilutions and house-keeping genes in the other plate also?

5. If I want to use delta-delta CT (comparative CT method) method (which I read somewhere is the method of choice for microarray validation), do I have to perform the validation experiment (i.e checking whether the efficiencies of target and reference/house-keeping gene are approximately equal) for all my targets (~ 20 genes)?

I think this is it for now.

Please provide you suggestions...

Thanks a lot,

Kart.





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