[DIYbio] What's wrong w/ qPCR? WAS:qPCR fluorescence detection dynamic range

On Tue, May 21, 2013 at 6:14 PM, Josiah Zayner <josiah.zayner@gmail.com> wrote:
> People don't _not_ do Real Time PCR because the equipment is so expensive.
> They don't do Real Time PCR because it is not very reliable.
> Intra-experiment variability is very high. Protocols are complicated and all
> the reagents are expensive.
>

You have some points there. A qpcr machine + reagents is expensive.
I'm not sure what you mean by complicated protocols, but developing
and fine-tuning an assay is quiet difficult to get it right. Once you
have it developed, it doesn't show that variability you describe. I
repeated qPCR results and there is low variability. It's not perfect
and there are some inconsistencies sometimes.

> With RNA quality being a hugely important factor most home labs are not
> equipped to do anything successful.
> Real Time PCR protocols that do more then detect copy number of a gene are
> complicated and require preventing DNA and RNAse contamination. You need to

What do you mean by "do more then detect copy number of a gene"? Our
company developed assays that can detect drug resistant mutations in
highly polymorphic regions using melt curve analysis. The advantage is
that I can get results from low copy fragments and from low mutant:WT
ratios (much more WT than mutant).

I also had samples sequenced to compare with our qPCR assay. The
problem so far is computational and time-consuming. Computational
meaning the seq files only show a single base-pair, even if the
samples are mixed WT/mutant. My way around it was looking at the
chromatogram and looking for dual peaks in mutation regions.
Time-wise, sending for sequencing gives me results in about 1-2 days.
A qPCR assay takes at most 2 hours to run on the machine. From
purification to qPCR to analysis for a sample, this might take 6
hours.

> With Sequencing/Deep Sequencing starting to become really cheap and you get
> to see the copy number of every transcript not just the ones you PCR it is
> becoming the goto technique.

How cheap are they? Just wondering what the cost comparisons are. In
developing our assays, I had to test against standards like
line-probe-assays, which are pretty expensive per sample.

>
> Most Real Time PCR used nowadays is for diagnostic stuff.
>

That's true. May I ask what other uses people have been trying to
apply qPCR to are? I mean, diagnostic assays are just the end product
and utilize the power of qPCR for useful work.

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