Question

10. You performed an RT-qPCR experiment to get the following Cq values:

Ct values	Before food	1h after food	2 h after food	3 h after food
Fat gene	23	24	22	24
GAPDH	25	26	27	26
DCq	2	2	5	2
DDCq		0	3	0
Fold change

Calculate the DCq, DDCq, and fold change numbers; express fold change numbers as N fold increase or M fold decrease rather than as fractions. What conclusions can you make about expression of the fat gene and the effects of eating? What assumptions do you make in using the DDCq method?

Answer 1

Expression in fold change is calculated as 2^-ΔΔCt (DDCq in your table)

Before food would be your 1

1h= 2^-0 = 1

2h= 2^-3=0,125

3h=2^-0= 1

What conclusions can you make about expression of the fat gene and the effects of eating? These results are not so conclusive and there are no evident effects rather than a downregulation at 2h.

The Delta-Delta-Ct (ddCt) Algorithm is an approximation method to determine relative gene expression with quantitative real-time PCR (qRT-PCR) experiments. Compared to other approaches, it requires no standard curve for each primer-target pair, therefore reducing the working load and yet returning accurate enough results as long as the assumptions of the amplification efficiency hold.

1. Take the average of the Ct values for the housekeeping gene and the gene being tested in the experimental and control conditions, returning 4 values. The 4 values are Gene being Tested Experimental (TE), Gene being Tested Control (TC), Housekeeping Gene Experimental (HE), and Housekeeping Gene Control (HC).
2. Calculate the differences between TE and HE (TE-HE) and TC and HC (TC-HC). These are your ΔCt values for the experimental (ΔCTE) and control (ΔCTC) conditions, respectively.
3. Then, calculate the difference between ΔCTE and ΔCTC (ΔCTE-ΔCTC) to arrive at the Double Delta Ct Value (ΔΔCt).
4. Since all calculations are in logarithm base 2, every time there is twice as much DNA, your Ct values decrease by 1 and will not halve. You need to calculate the value of 2^-ΔΔCt to get the expression fold change.