Exponent negative equals to negative result in real time PCR

22 June, 2011, I went to meet a customer complaining that their real time PCR results were not consistent over the week, where the positive result in one run becames negative in the next run, with the same sample (s).

Before I went, I got some run files from her.

Back in the office, I noticed that there was nothing wrong with the result (s), except that there are some minor differences in her replicates, which could be pipetting error.

To my surprise, after having some discussion with her on how to analyze her result (s), she told me that the way she call a sample as positive or negative based on the exponent from result in the Result column generated by the software; i.e, 2.223e+1 is positive, whereas 2.223e-0.1 is negative.

OMG, she is analyzed her result based on the exponents of the quantitative results generated based on the standard curve she had.

I ended out discussing with her what is the meaning of exponent, and how to interpret the result.

Wow, this is the most creative way of analysis a real time PCR result so far I met.

company's propriety issue?

Recently, in order to reduce the time and effort to design and optimized an real-time PCR or qPCR assay, lots of scientist opts for pre-designed assay from certain company (s).

These assays are indeed helps a lot of scientist by reducing their time and effort to design their own assay where they need to consider the primer (s) and/ or probe (s) specificity and efficiency, mispriming and etc.

However, one of the common issues faced by the scientist when they wish to publish their data using these assays is due to the company's propriety issue, the sequence information for the primer (s) and probe (s) can not be disclosed. But these information is critical as it is required by MIQE guidelines.

So now, does these companies still want to keep these information to themselves, which might affect the sales of these assay (in my opinion) or just do the customer a favor by disclosing these information so that there are more citation for these assay in near future??????

pyrosequencing and PCR

I made a mistake by answering "Yes" to this question, and probably I will say "No" if I have think twice to it.

The question is "When you run the agarose gel, you get only a single sharp band; does this means that your pyrosequencing will be fine?"

OMG, I shouldn't have answered "Yes", but should be a firm "No" or just a more polite "Probably".

The issue here is, before subjecting a DNA sample for pyrosequencing, it needs to be denatured into single-stranded DNA after the PCR. And to get the biotinylated PCR product to become a single-stranded DNA, we need to captured or immobilized the biotinylated PCR product to sepharose beads, which will be then denatured and washed, and finally been released into a plate which contains the buffer that has all the other component (s) for pyrosequencing reaction.

Of course, without a good PCR product, you will not get any pyrosequencing result, but by having a good PCR product will not promise you to have a great pyrosequencing product or result as well, because as you can see, there are so many steps are taken place after PCR which could go wrong if you are not careful.

So, the answer to the question "When you run the agarose gel, you get only a single sharp band; does this means that your pyrosequencing will be fine?" should always be a firm "No".

RT-PCR and/ or qRT-PCR

What will you suspect when you get a negative expression of your gene of interest but a positive expression of your housekeeping gene? Furthermore, maybe you wish to try to get a hint if your primer set is working fine by amplifying the gene of interest with a DNA sample instead of a RNA sample, and yes, it amplifies.......

So, what could go wrong here?

• Poor RNA quality? May be. But should be checked prior to any downstream assay, if possible.
• Not optimized primer design? Could be.
• Not optimized RT-PCR or qRT-PCR condition? Perhaps. I still remember when I used to amplify low abundant gene (s), and just by increasing the number of cycle from 40 to 50 cycles, all the genes got amplified.

Or the gene is not expressed in the sample then...... Worth to get this investigated, I guess, in my opinion.

a better data analysis method???

Lots of people will be more concern in data analysis of their qRT-PCR result and will straight away ask me which data analysis method is better than the other so that they can use for their studies. I encountered this question (s) a lot during the installation and training of real time PCR.

However, in most of the case, I found out that they don't even know of or consider the essential of starting material/ sample quality, how to process their samples to get a pure and intact nucleic acids, the quality of their extracted and purified nucleic acids etc etc. These could detrimentally effect the reliability and quality of their data.

And of course, not to mention on the experimental design. Another area which lots of people or researcher (s) overlook its important.

lyophilized primers, concentration.....

Normally primers for PCR are shipped in lyophilized format, and prior to use, these primers need to be dissolved in TE buffer to provide a stock solution concentration of 100 µM.

Wondering have you ever measured the concentration of primers' stock solution with spectrophotometer???

If not, how sure are you that the concentration is accurate???