5 replies to this topic

[kedar]

Dear all,

The story goes like this:
I ordered taqman gene expression assay kit. I am trying to check the expression of some candidate genes from microarray.
Say a protein ''cathepsin L''
1. I use 5 µl of cDNA from a sample+20 µl mastermix (includes everything) and amplify the cDNA in realtime pcr. After the pcr, I take this PCR product as 10^10 copies/5 µl (arbitrarily assigned) and dilute it 10^9, 10^8, 10^7......10^2 ....Now, I use these standards and again run a PCR (realtime) and check the amplication.....some samples are outliers, few saturated and others, generally are fine (amplifying at 3.. cycle)....I prepare a standard curve from the samples that were perfectly fine (giving .999 r value, slope 3.3...)
So, the question is CAN I USE THIS (let's say 10^10 sample) as a standard from now on? ....meaning i dilute it and prepare the standards for next pcr run. and freeze the product at -20...

GENErally, i see people complain about contamination etc...the problem that i am facing with this easy method is when i repeated the realtime pcr after 1-2 weeks using 10^10 sample and diluting it...After i ran the pcr, i realise the product is degraded although it was at -25.......IS THIS BY CHANCE? or the pcr product cannot be stored at all!


Appreciate your response,
Kedar Ghimire

[pcrman]

I don't understand why you want to reamplify your PCR product.

[Rsm]

It seems he wants to use the PCR product as an external standard. Like a spiked-in RNA... I wouldn`t do it, because any nuclease activity would result in less amplification. Better use a circular vector, those are more resilient to nucleases.
Anyway, using a PCR product does not tell you about the reverse transcriptase efficiany, so if you want to check mRNA levels, better use an external mRNA (like plant mRNA or whatsoever).
Cheers,
Minna

[kedar]

Hi both,

thanks for the reply.

Yes, I am talking about external standard for the target gene. If i want to know the expression levels of cathepsinL in my samples, I must have the standard curve from a known sample to compare the values with my unknown samples, isn't it?

I amplified the pcr product just to get enough amount to be used again as standard for the next time.

Instead of circular vectors (you need to clone etc); i was asking whether simply this pcr product can be used to prepare a standard curve by preparing a serial dilution.
1. Is this valid?

Obviously, as Minna said the product degrades soon. So, for long-term, i feel it's better to clone and prepare plasmids etc...

I hope you are more clear about my query now..

thanks
Kedar

It seems he wants to use the PCR product as an external standard. Like a spiked-in RNA... I wouldn`t do it, because any nuclease activity would result in less amplification. Better use a circular vector, those are more resilient to nucleases.
Anyway, using a PCR product does not tell you about the reverse transcriptase efficiany, so if you want to check mRNA levels, better use an external mRNA (like plant mRNA or whatsoever).
Cheers,
Minna

[Rsm]

>1. Is this valid?
No.

1. Your standard will degrade.
2. You can not compare normal PCR with RT-PCR.

Minna

[molgen]

It's not the best thing but, yes you can use it. It's a poor mans solution.
Over time your standard will degrade as minna pointed out.
The efficiency of PCR is different from TR-PCR.


If you do all your samples in one go and use freshly diluted standard curve you can do it.
You can try pooling 1-2µl from each RT and use that as a standard.