Team:KU Leuven/Project/Glucosemodel/qPCR

Why qPCR?

We performed this qPCR for two main reasons:

1. With a qPCR we can check if our genes of interest are properly transcribed. This is a good characterisation of the methyl salicylate brick (BBa_K1060003).
2. We would like to know the amount of transcripts as an input for our methylsalicylate model. Because the mRNA production step is responsible for a large part of uncertainty in our model we would like to bypass it. In order to attain our goal without needing the transcription rates we tried to determine the in vivo mRNA concentrations using qPCR.

The notebook on the qPCR experiments can be found in the journal.

Sample Preparation

We first started with the sample preparation, as described in the qPCR protocol . For this experiment we used our regular E. coli strains (DH5α), harbouring our methyl salicylate brick and used 3 biological repeats.
After preparing three samples we started with the isolation of RNA. In a next step we removed DNA contamination by doing an extra DNase (Turbo DNase, Ambion) step as described in our protocol.
We then used the Nanodrop to get to know the concentration of our RNA and got the following results:

These results are satisfactory. Only the 260/230 values are low because of the high salt level of the DNase buffer. After purification these values will rise.

Troubleshooting

Thanks to some brainwork we got to a hypothesis that could explain this problem. Because we used a high copy number plasmid (ORI: pMB1), the proportion of plasmid DNA is far higher than the proportion of genomic DNA. The manufacturer advised to use a more rigorous treatment, but could not assure us that all traces of plasmid DNA will be removed because we are using a high copy number plasmid. So we had to find another way around of this problem.
After advice from dr. Basak Ozturk we decided to go for a restriction digest on the RNA samples. We figured that we’d best cut it with an enzyme that cuts the fragments that will be amplified in the qPCR itself. On this way the contaminating DNA will not be amplified while the cDNA will be correctly amplified and we could take unbiased conclusions.So we went looking for a good restriction enzyme that does the job perfectly for our two genes of interest.

New hopes crushed

We came to the restriction enzyme MvaI. This should cut our plasmid on the right places as shown on figure 3.
The MvaI restriction enzyme should cut inside both of the qPCR amplified regions (the regions between the “blue boxes”), so that only linear amplification is possible there and that interference should be eliminated.
Unfortunately, after running some PCR’s and DNA electrophoresis gels to check whether we finally got rid of our contaminating DNA, we still had a disappointing result (see figure 4).
It might not be clear on the images but all our samples still show bands which means that our digestion-trick did not work.
This means that we are unable to remove the contaminating plasmid DNA. As previously mentioned we think that is due to the fact that the proportion of plasmid DNA is far higher than the proportion of genomic DNA, as we used a high copy number plasmid. This higher proportion of plasmid DNA is very hard to remove using DNase treatment. If we really want to go on with the qPCR, the only option is to clone our genes of interest in a low copy number vector or into the genome of our host strain. Since we were already quite busy with cloning, we decided to end the qPCR adventure here.

Figure 4 | Drawing that shows the MvaI restriction sites. The blue boxes show the primers we designed for the qPCR.

Figure 5 | DNA gel electrophoresis after two different PCR reactions where each amplifies a different region on our plasmid.

Conclusion

We decided to publish this story (even though it was unsuccesful) to warn the other teams for the difficulties that come with the removal of contaminating plasmid DNA in a RNA sample preparation.