PCR006: Obtaining standard curves for L452R, K417T and E484K strains. (open)

This is a copy of the original notebook created by Trevor Ho and Nahuel Moreno in Nick Gilbert group (https://chromatinlab.org/) for the COVID Wastewater Scotland project (https://biordm.github.io/COVID-Wastewater-Scotland/)

We have obtained RNA from our G-block plasmids through qPCR in ​Inaccessible Entry and will be testing what range of RNA concentrations we can accurately measure.

Contrary to ​Inaccessible Entry we are only interested in measuring the unspecific binding of the FAM and NED probes in order to decide if we have to reorder them or not. qPCR experiments should be wrapped up soon.

For simplifying the test and reducing the number of experiments we will only test WT probe on MUT template and viceversa. If either binds to the template and we obtain concentration-dependant signal we know the probe is bad and we will have to reorder it.

Ideally we will look for one working pair of specific probes which will allow us to make a test in waste water samples.

We will be testing the binding of pairs of probes using our RNA obtained in ​Inaccessible Entry.

For this we will use a range of concentrations of RNA from the L452R, K417T and E484K strains and the Takara one step RT-qPCR kit. This is the kit we have in highest abundance and it is the fastest. In terms of efficiency, we have not seen any significant differences between kits (​Inaccessible Entry )

Plate layout:

Our RNA from ​Inaccessible Entry turned out to be low concentration so we couldn't dilute it to a final concentration of 1 μg/μL. We have four of the samples at 500 ng/μL and two at 100 ng/μL. We thereafter performed serial dilutions for obtaining the desired concentrations of sample.

Initial volume of RNA is pipetted from stock, subsequent volumes are pipetted from the prior dilution tube.

We will only have three concentrations in order to maximize the sample and probe coverage.

The PCR is assembled as per specifications of the Takara SuperScript RT-PCR kit.

There will be reactions with six different probes and each will be run in their corresponding WT and MUT strains.

Mastermix recipe for each probe pair (16 wells each):

We eadded the diluted RNA sample into each well (1 μL) and then pipetted 9 μL of mastermix into each corresponding well. Mixed all the wells by pipetting.

PCR run as the Takara SuperScript kit specifications.

L452 shows no differentiation in binding of mutant and wildtype probes. Both probes bind with equal affinity to both templates.

E484 and K417T show high specificity of the WT probe (FAM, orange crosses) and unspecificity in the mutant probe (NED, blue dots). However, for both mutant probes, it could be possible that the probe binds with higher specificity to the mutant template, which is visible in the more gentle lines in the WT template signal compared to the MUT signal. This could mean that the MUT probe could potentially be outcompeted in a competitive assay and this set of probes can be used as a method for quantifying the specific strains.

This experiment will be followed up in Stilla ddPCR.

This experiment is now closed.