Hello everyone,

I am extremely confused about analysis of qPCR data after virus infection or stimulations, and I need help. I hope I can explain properly.

Let's say we have two different cell lines.

1. HFF WT and

2. HFF protein A knockout.

We infect (or stimulate) these cells with virus and then do IFNB1 and GAPDH qPCR.

So, we will have these conditions:

• Mock HFF WT
• Virus-infected HFF WT
• Mock HFF protein A KO
• Virus-infected protein A KO

Let me try to explain what I understand how people analyse this kind of data:

First, you get the Cq values of IFNB1 and GAPDH.

Then, you do (IFNB1 - GAPDH)

Using (IFNB1-GAPDH) values, then you do (Virus infected HFF WT- Mock HFF WT) and (Virus infected HFF protein A KO - Mock HFF protein A KO).

Then you are doing POWER(2; -(Virus infected HFF WT- Mock HFF WT))

or POWER(2; -(Virus infected HFF protein A KO - Mock HFF protein A KO).

My problem is:

Since the mock cells are not stimulated, they usually give either negative or very high Cq value of IFNB1. (for example: 36, 38, 39). And this value is totally randomly changes experiment to experiment. Sometimes Mock HFF WT is 36, and the Mock HFF protein A KO negative. Sometimes vice versa. These also changes between biological replicates all the time. (I usually write 40 when it is negative, which is my cycle number).

In this case, can I just consider all the mock cells will have negative value and consider their Cq is 40 (eventhough I get 35-39). Of course they can have some IFNB1 even they are not stimulated/infected and this can change between these two cell lines, but having different mock results changes the whole outcome.

Note:

My IFNB1 Cq values are usually 20-26 in infected cells.

My GAPDH Cq values are usually 20-25.

I use GoTaq.

I convert total of 400 ng of RNA to cDNA (in 20 uL).

Then I take 1 uL for qPCR, so 20 ng. Can increasing the cDNA amount help me to get positive values from Mocks and equalize them?

Thank you very much.