First I need to export the data from BioRad CFX manager.
- Click Export
- Go to Export All Data Sheets
- Select CSV as export file type
This causes a ton of CSV's to be produced most of them have absolutely no value for PCR Miner. The one we need is titled: Quantification Amplification Results _ FAM. FAM denotes the luminescence frequency which picked up the lumination from the SYBR green dye.
Now we need to format this file.
- Rename each column with the appropriate Gene, Replicate, and sample Number.
Now you just need to copy the data into the entry area of PCR Miner.
PCR Miner pops out the data which you will download from the following screen:
Then this text file can be opened in Excel. Once in excel I highlighted the Cycle threshold value and the efficiency for each population. Then I created the expression value with the equation =1/(1+Efficiency)^CycleThreshold. I did this with both the TLR2.1 and Actin genes. Finally I divided the TLR2.1 expression values by the Actin expression values to get the normalized expression value of TLR2.1.
This is just the first full run using 8 samples per population per treatment but we can see some interesting trends. Expression of TLR2.1 is nearly doubled in the Fidalgo population (N) and tripled in the Oyster Bay population (S) due to Heat Stress. The values for the Dabob population are a little dubious due to the lack of expression from about half the population. Though it does show that while expression seems to be down regulated, it is roughly the same between control and treatment.
Very cool - would be great to see individual data points, violin? plots, something similar to size at spawning of olys?
ReplyDeleteCouple of things:
ReplyDelete1. Make sure when you export the data from CFX (and/or the Opticon) that you've made sure that "background subtraction" is OFF.
2. Taking a very quick glance at the data, I noticed that the "AverageCT_OfReplicateSamples:" calculated for TLR2.1_HC
by PCR Miner is inaccurate (sort of). More than half of the individuals for that group did not produce a CT value at all. It looks like PCR Miner calculated the "AverageCT_OfReplicateSamples:" from just those samples that actually produced a CT value. This means that using that value is not an accurate representation of TLR2.1 expression of that group.
This is a long-standing issue that we've grappled with in the past. It's been so long since we've done serious qPCR expression analysis that I can't remember how we dealt with null values for amplification. Before going further with additional analysis, you should definitely discuss with Steven.