Today Sam and I took a look at the qPCR data from last week’s trial run. We found that the third primer we used (CYP1A) is not functional, as there was no amplification in wells that had the CYP1A primer. Elongation factor was supposed to be our normalizing gene target, but we found there to be a relatively high range in Cq values for the elongation factor gene across treatments (23.92–32.97). This may be due to an outlier cell (1H) which could have been caused by a pipetting error or sample contamination. For future qPCR runs, we will include technical replicates to increase the reliability of the data (we decided not to use technical replicates for this round as it was only a practice run).
Curves for the HSP90 primers looked good and melt curves for elongation factor and HSP90 indicated that there was only one amplification product in each well. We also reviewed some of the basics of qPCR, including how the curve threshold is determined, melt curves, etc.
Link to qPCR data: http://owl.fish.washington.edu/scaphapoda/ronit/admin_2018-09-12%2015-40-46_BR006896.pcrd
Notes about well setup:
1A–1D: Control treatment, elongation factor primer
1E–1H: Row 8 Column 1: CO2 treatment, elongation factor primer
2A–2D: Control treatment, HSP90 primer
2E–2H: CO2 treatment, HSP90 primer
3A–3D: Control treatment, CYP1A primer
3E–3H: CO2 treatment, CYP1A primer
4A–4C: Negative controls (no cDNA)