Hi Laurence Stuart Hall,

I faced the similar problem during my qPCR validation of some genes having very low copy number. I was validating few of the genes which show low fold changes in my microarray data but are important for biological interpretation.  I performed relative quantitation of the GOI with a preclude standard curve with which Ct values were compared to find the relative abundance. My validation data matches very well with the microarray data if the test Ct values are in the range of 15 to 25. Above Ct value of 25, there is much deviation among the 2 data. I did not find anything technically illegitimate in this. As per my knowledge technically there is no other ways than pre amplification before actual PCR.

Hi Laurence Stuart Hall,

I faced the similar problem during my qPCR validation of some genes having very low copy number. I was validating few of the genes which show low fold changes in my microarray data but are important for biological interpretation.  I performed relative quantitation of the GOI with a preclude standard curve with which Ct values were compared to find the relative abundance. My validation data matches very well with the microarray data if the test Ct values are in the range of 15 to 25. Above Ct value of 25, there is much deviation among the 2 data. I did not find anything technically illegitimate in this. As per my knowledge technically there is no other ways than pre amplification before actual PCR.

Hi,

You most likely know this but try using an alternative cDNA source that you know the GOI is expressed high to avoid the big CT values. At least then your standard curve will be there.

Asif 

Hi Asif

yes I will do that exemplified by my housekeeper which will be used to normalise the data but the problem is how to contruct a standard curve from my gene of interest which we know is expressed at very low levels; so even with neat cDNA the Ct values are very high which raises the question: How do you dilute the cDNA to construct a standard curve for this gene of interest and if you cannot how can you quantitate this gene by qPCR; Perhaps you cannot without a valid standard curve ? Perhaps in the absence of such a curve a need to find another of quantitating this gene between 2 types of sample

Thanks Anja

pre amplification apparently can work but is very expensive and RT with a gene specific primer might also be an option

I don’t understand why do you need to do a standard curve? Most people accept normalization to a housekeeping gene. It’s not perfect but acceptable. The turnovers of your transcripts and the housekeeping transcripts are not the same…Moreover, most classical housekeeping genes people use have several dozens of pseudogenes that are transcribed too.

If it’s really necessary for you to do a standard curve then go the other way. I mean increase your input sample by 2, 3, 4 or as you whish

Dear Laurence,

It is trick to deal with the low copy number genes. But if you don't need to worry about the  quantity of  materials  you can get, you certainly can increase the starting material i.e RNA for the treverse ranscription.

There are two quantitative methods you can use. Relative quantitation, which engages one or a few reference genes along your target gene, in your case, you will need to find a house keeping gene which expresses at low levels in order to accurately reflect the true value of your target gene expression. By doing so, you will still need to compare  and optimize the amplification efficiency for both of your target gene and reference gene to siminar level.

Second method is absolutely quatinfication, which appears that you are using above, in this case, you will need to do standard curve. You will have to increase your target material dramatically,  for instance, you may using 1ng of RNA right now, instead, you can use ug of RNA to do the reverse transcribe step, that way you can make sure you have enough copies in your cDNA pool to be detected. every 100 times more of starting material will bring  around 7 ct down.

Good luck

Lingli