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relative quantitation problem plz help


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#1 chirpy

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Posted 19 March 2009 - 10:26 PM

hi all,

I am in a gr8 trouble and need help. Actually i have to double check my Differential Display data with Real time. I have got certain sequences differentially expressed by Differential Display. I blast and got name of genes. Now, i hv to check expression of these genes in my different age samples. I did by RT-PCR but what is troubling me is my Hsk gene beta-GUS is showing a difference of mean Ct values three between two samples. replicates are tight. i dont know whether it is significant or normal to happen. when i use these values for ^^Ct calculations then certain genes expression pattern correlate with my data, while certain do not. In one case my one gene show R value of 200, that too in opposite age group.
Another thing is that in Differential display the relative intensity of bands corresponds to level of mRNA species directly, it is not normalized to any housekeeping gene. Is that the reason my data not correlating? Please help.

#2 littleaxt

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Posted 19 March 2009 - 11:51 PM

Hi

I don't know anything about Differential Display, sorry. But please keep in mind, that there is no general housekeeping gene that is suitable for every type of cell or experiment. Have you validated beta-GUS or has someone else done it using the same experiment like you? Maybe beta-GUS is just a poor reference in your case and you should find another reference gene or even better genes.

Good luck and all the best!

#3 chirpy

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Posted 20 March 2009 - 09:43 AM

Hi

I don't know anything about Differential Display, sorry. But please keep in mind, that there is no general housekeeping gene that is suitable for every type of cell or experiment. Have you validated beta-GUS or has someone else done it using the same experiment like you? Maybe beta-GUS is just a poor reference in your case and you should find another reference gene or even better genes.

Good luck and all the best!


hi littleaxt,
Thanks 4 ur reply. can u plz. tell me how shd i validate my housekeeping gene ? actually one of my seniors had used B-gus many yrs. ago in her study. so i went 4 same. can the house keeping gene change with age of animal like i mean i m comparing b/w two age groups. is it o.k. to think that my young age cell synthezises more basal level of all mRNA compared to my old age group.
therefore, variation in housekeeping gene is there. or this is wrong ?

#4 littleaxt

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Posted 23 March 2009 - 02:36 AM

Hi,

the most common thing is to compare several potential reference genes with each other. There are different approaches, how to compare the obtained data (e.g. Vandesompele et al 2002, Lindbjerg et al. 2004, Pfaffl et al. 2004, Szabo et al 2004... more info see http://normalisation...fication.info/). To decide what's best is up to you. I used three different approaches and compared them and lucky me I they came up with almost identical results. Check out for other paper where people validated their reference genes, there is a lot to find. But of course this means reading, reading, reading... No way to circumvent this.
If you lack of information about potential reference genes (working with a non-model organism) you should test your gene against a artifical RNA-spike and normalize against amount of samples cells or amount of RNA. That's not perfect, but better than nothing.

I come from another field of biology and I'm more or less just a "user" of qPCR, but as far as I understand it is nowadays absolutly important to use validated reference genes and if no one has done it for you experiment and your orgnaism so far, you have no chance to avoid it doing it yourself. There is lots of information about this circulating around.

Hm, can't tell you much about cell biology and find it sometimes hard enough to deal with this molbiol. But I suffer a bit from the same problem. I have in-situ samples, lacking information about age or physiological state of the animal. I therefore expected also higher variance in my reference genes as I assumed the expression profile to be different between a young an d an old animal. So I chose samples blind and searched for a stable genes not affected by age or physiological state.
It's ok, to assume that young cells have a higher basic level of mRNA than older ones, but I would prefer to know it ;-). Therefore test it!!!
But maybe a cell biologist can asure you that this is assumption is right, I can't and would prefer to be on the safe site. Just image you run your whole experiment and in the end a reviewer insist on the proof that they do not differ. Weeks of work for nothing, worst case...

All the best!

#5 chirpy

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Posted 25 March 2009 - 03:07 AM

hi,

thanks a lot for your suggestions. it is really helpful to know all this info. yes, i agree with you that a perfect housekeping gene is must. so i will be looking out now for more options. good luck to you as well for your experiments.

thanks for help.





Hi,

the most common thing is to compare several potential reference genes with each other. There are different approaches, how to compare the obtained data (e.g. Vandesompele et al 2002, Lindbjerg et al. 2004, Pfaffl et al. 2004, Szabo et al 2004... more info see http://normalisation...fication.info/). To decide what's best is up to you. I used three different approaches and compared them and lucky me I they came up with almost identical results. Check out for other paper where people validated their reference genes, there is a lot to find. But of course this means reading, reading, reading... No way to circumvent this.
If you lack of information about potential reference genes (working with a non-model organism) you should test your gene against a artifical RNA-spike and normalize against amount of samples cells or amount of RNA. That's not perfect, but better than nothing.

I come from another field of biology and I'm more or less just a "user" of qPCR, but as far as I understand it is nowadays absolutly important to use validated reference genes and if no one has done it for you experiment and your orgnaism so far, you have no chance to avoid it doing it yourself. There is lots of information about this circulating around.

Hm, can't tell you much about cell biology and find it sometimes hard enough to deal with this molbiol. But I suffer a bit from the same problem. I have in-situ samples, lacking information about age or physiological state of the animal. I therefore expected also higher variance in my reference genes as I assumed the expression profile to be different between a young an d an old animal. So I chose samples blind and searched for a stable genes not affected by age or physiological state.
It's ok, to assume that young cells have a higher basic level of mRNA than older ones, but I would prefer to know it ;-). Therefore test it!!!
But maybe a cell biologist can asure you that this is assumption is right, I can't and would prefer to be on the safe site. Just image you run your whole experiment and in the end a reviewer insist on the proof that they do not differ. Weeks of work for nothing, worst case...

All the best!






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