Sam's Notebook

qPCR Analysis - C.gigas Matt George Poly:IC Diploids

by Sam White ~1 min read

Ran a “quick and dirty” qPCR analysis for the qPCR’s I’d previously run:

All analyses are documented in the following R Project:

Here’s a brief overview of what was done:

  1. Select only diploid samples (currently, only have control/injected groups for diploids - not triploids)
  2. Normalize to actin by calculating delta Cq.
    • This was done for the sake of time. Actin and GAPDH (the other normalizing gene run) shows evidence of a treatment effect. Actin was selected over GAPDH due to smaller range between mean Cqs of Control/Injected.
  3. Run t-test to determine p-values between control/injected withing each actin-normalized gene.
  4. Box plot all delta Cq values.
  5. Calculate 2^(-delta delta Cq) (subtract Control delta Cq from Injected delta Cq) to determine fold change in expression for each gene.
  6. Plot fold change in expression as bar plots. Values >1 indicate increase in relative expression. Values <1 indicate decrease in relative expression.

RESULTS

Box Plots (delta Cq)

Box plots of delta Cq (i.e. normalize to Actin). T-test identified only a single gene (marked with orange asterisk) as significantly different between Control/Injected: DICER

boxplots comparing 6 different genes - orange asterisk above Cg_DICER indicates statistically different between Control and Injected

Fold Change (2-(ddCq))

Fold change in gene expression of Injected individuals, relative to control samples.

Bar plots showing fold change for each gene. Value > 1 means "upregulated"; value < 1 means "downregulated".