Health sciences and medicine

Real time PCR report

Calculation:

? ??Ct = ?Ct (treated sample) – ?Ct (untreated sample)
Where,
? ?Ct (sample) = Ct (target) – Ct (endogenous control), therefore
? ??Ct = (Ct (target, untreated) – Ct (endo ctrl, untreated)) – (Ct (target, treated) – Ct (endo ctrl, treated))
Where,
? Ct (target, untreated) = Ct value of target (MMP1) in the untreated sample
? Ct (endo ctrl, untreated) = Ct value of endo ctrl (18S) in the untreated sample
? Ct (target, treated) = Ct value of target (MMP1) in the treated sample
? Ct (endo ctrl, treated) = Ct value of endo ctrl (18S) in the treated sample
? Can now calculate the ratio of target gene in our untreated relative to our treated by taking 2??Ct

Example:

Mean Ct values Cell line 1 Cell line 2
Endo ctrl (SNORD48) 16.170 15.895
Target gene (miR-31) 21.225 19.763

? ??Ct = (Ct (target, untreated) – Ct (endo ctrl, untreated)) – (Ct (target, treated) – Ct (endo ctrl, treated))
??Ct = (21.225 – 16.170) – (19.763 – 15.895)
??Ct = (5.055) – (3.868)
??Ct = 1.187
2??Ct = 21.187 = 2.277

Target gene is upregulated by 2.277 fold in the treated sample compared with untreated control

The report should include a brief overview of the malignant pleural mesothelioma lines used and the qPCR technique, a calculation of the relative quantity (RQ) of miR-31 in sample 1 versus sample 2 (showing how the value was calculated and including a logical interpretation of the value) and a discussion of the advantages and disadvantages of the technique.

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