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Real Time PCR Normalization


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#1 sssbio

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Posted 30 May 2010 - 05:18 AM

Hi,

I need your help regarding quantification of mRNA levels of trageted genes in control and treated samples. I have no choice of reference housekeeping genes to be used for relative quantification as these genes are showing significant down regulation in treated samples. So what are other methods that I can use to quantify mRNA levels?

I will be very happy if you guide me for this.

Thanks

#2 lsek

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Posted 01 June 2010 - 10:04 AM

Hi,

You said that housekeeping genes are showing significant down-regulation in treated samples. How did you derive at such conclusion and what reference gene was used for normalization of such observation?

If the above observation is good, you could use the reference gene as mentioned above for your normalization.

In addition, an abundant reference gene that you could use is 18S ribosomal transcript (but must use random primers for 1st strand synthesis), Probably this reference gene will not be affected by the treatment.

Hope this help.

Cheers,

===><===


#3 sssbio

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Posted 07 June 2010 - 06:01 AM

Hi,

You said that housekeeping genes are showing significant down-regulation in treated samples. How did you derive at such conclusion and what reference gene was used for normalization of such observation?

If the above observation is good, you could use the reference gene as mentioned above for your normalization.

In addition, an abundant reference gene that you could use is 18S ribosomal transcript (but must use random primers for 1st strand synthesis), Probably this reference gene will not be affected by the treatment.

Hope this help.

Cheers,


Hi,

Thanks for your suggestion.

Actually, I did reverse transcription (1000 ng RNA) and single stranded cDNA (50 ng) used as template for PCR with housekeeping gene specific primers. I did PCR for only 20 cycles and load on agarose gel. I found that actin, tubulin, ribosomal protein L21 and GAPDH genes significantly showed very low intensity band amplification in treatment.

Then I used 18S primers and they worked very well in both control and treatment.

Thanks for your kind reply.

Sincerely

sss

#4 phz@nebion.com

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Posted 06 October 2011 - 11:17 AM

Did you try using RefGenes to find suitable reference genes? It's a free online tool that processes data from thousands of microarrays to find genes that are stable for chosen tissues or conditions. RefGenes is available at www.refgenes.org




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