MBIO 4612 Lecture 3: Eukaryotes Lecture 3
9 Feb 2020
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Calculates relative gene expression data while accounting for differences in primer efficiencies. Derived from northern blot used reference genes to estimate changes in mrna levels. Ct = cycle threshold ct = control - treated. Primer efficiency (e) for goi and hkg. Efficiency decreases with every additional cycle affects how much product is made and the ct values! Materials start to be used up or lose activity. 80% = 1. 8 in calculations not 0. 8, etc! Calculates change in relative fold gene expression between 2 samples doesn"t consider primer efficiency. Compares change in ratio of target gene to housekeeping gene: if ratio goes up expression of target gene has gone up (hkg should be stable) Comparing to hkg normalizes the data ensures change is due to gene expression not different conditions (i. e. more rna in 1 sample) Make sure your exponent in fold change is positive! If ct is positive, don"t use negative in exponent.
