After testing out the RPL5 and TIF3s6b v2 and v3 primers yesterday on pooled cDNA, we determined the primers looked good, so will go forward testing them on a set of P.generosa hemolymph cDNA made by Kaitlyn on 20200212. This will evaluate whether or not these can be utilized as normalizing genes for subsequent gene expression analyses.
Primers used:
| SRID | Primer_Name |
|---|---|
| 1787 | RPL5_v2_FWD |
| 1786 | RPL5_v2_REV |
| 1785 | RPL5_v3_FWD |
| 1784 | RPL5_v3_REV |
| 1783 | TIF3s6b_v2_FWD |
| 1782 | TIF3s6b_v2_REV |
| 1781 | TIF3s6b_v3_FWD |
| 1780 | TIF3s6b_v3_REV |
I used geoduck gDNA (162ng/uL; from 20170105) as a positive control.
Master mix calcs are here:
- 200200825_qPCR_geoduck_RPL5-v2-v3_TIF2s6b-v2-v3 (Google Sheet)
All qPCR reactions were run in duplicate. See qPCR Report (Results section below) for plate layout, cycling params, etc.
NOTE: These qPCRs used the remainder of all the samples.
RESULTS
These primers are not going to be useful as normalizing genes:
RPL5 spread is way too wide for use as a normalizing gene (~10 Cqs)
TIF3s6b doesn’t amplify in most samples (however, it is very consistent in those that it does amplify…)
Data files and amplification/melt plots are below.
RPL5 qPCR Report (PDF):
RPL5 CFX Data File (PCRD):
RPL5 CFX Results File (CSV):
TIF3s6b qPCR Report (PDF):
TIF3s6b CFX Data File (PCRD):
TIF3s6b CFX Results File (CSV):
RPL5 v2
AMPLIFICATION PLOTS
MELT PLOTS
RPL5 v3
TIF3s6b v2
TIF3s6b v3
