Monday 3/21/2016 – Test qPCR Results
The results of the initial test run of the qPCR conducted last week for the Actin and CYP1A genes came back after using the same procedure posted up in the previous notebook. Both genes single-plexed came back with good amplification at the cycle timing that we were looking for.
In addition, the control wells did not amplify as expected indicating the DNAsing procedure and Reverse-transcription was completed correctly for these samples, and hopefully the rest of them.
However, the duplexed gene wells did not work correctly, with only the Actin wells significantly amplifying during the course of the qPCR.
Duplex, Actin in Green, CYP1A in Blue
There’s no guarantee as to what might have caused the duplexing to fail, whether it could have been a simple saturation of the DNA polymerase with Actin or interaction between the dyes I’m not sure (this is what testing plates are for!)
My plan for this week is to work on finishing up Reverse-transcribing the samples and get input on how to proceed with the qPCR. There’s the option of attempting to figure out the issue with the duplexing and correcting it if possible and optimizing that; or simply proceeding with single-plexing the gene targets. The latter might require a bit more work but will definitely get results if it goes as smoothly as this test run.
Saturday 3/12/2016 – Big Update
DNAsing has been finished for every sample without problems following the same procedure as found in the Common Lab Protocols, with the only deviation being 5 minutes total of DNAse inactivation as compared to the 2 minutes stated there. The next step of the project is going to be getting ready to reverse transcribe the samples I DNAsed and then getting the information necessary to qPCR it all! To sum it up…
Remaining qPCR Steps:
- Reverse Transcribe 6 DNAsed samples (following lab standard protocol) – Complete
- Run test plate to check primer/probes, multiplexing, and reverse transcription
- Reverse Transcribe remaining 30 samples
- Run the qPCR for B-actin and CYP1A on all 36 samples
- Repeat steps 2 and 4 for MTA and VTG gene if time allows (using B-actin as the reference)
Test Sample Run of qPCR
1. Thaw Master Mix and nuclease-free water at room temperature and then vortex for 5 seconds.
- Number of reactions is 81 6, additional reactions will make it 83 7 total reactions to ensure against pipetting error.
- Setup will be 10 uL of Master Mix, 1 uL each of Forward and Reverse Primer in addition to Hydrolysis Probe(s), 4 uL of Template DNA (depending on concentration of reverse transcription products), and 3 uL of Nuclease-Free Water for each reaction (also depending)
- Total for test run: 70uL of Master Mix, 7 uL of F/R Primers+Probe(s), 21 uL of Nuclease-Free Water with 16 uL of mix added to each well after being vortexed briefly to mix.
- Add 4 uL of diluted Template DNA (1 ug/ul, maybe 10 ng/ul?) to each well from the respective samples, seal the optical plate, and centrifuge briefly to collect components at bottom of the wells.
- No Template Controls will receive an additional 4 uL of Nuclease-Free Water, No RT Controls will receive uL of non-RT “RNA” Sample, No Amp. Controls will receive an additional 10 uL of Nuclease-Free Water to replace DNA polymerase found in Master Mix.
(Standard) Thermal Cycling Conditions
1 Cycle of: 95 Celsius for 2 minutes to activate GoTaq
40 Cycles of: 95 Celsius Denaturation for 15 seconds followed by 60 Celsius Annealing/Extension for 1 minute
This data is going to subject to change however and will be updated as necessary until it is ready to be ran through. I’m keeping a full data set table which includes all the data gathered from the samples up to this date here, Capstone Sample Data. Plan is tentatively going to be to discuss and make changes to the procedure early next week and then hopefully run the test to determine if multiplexing is going to be possible later next week.