Housekeeping genes - Cq values - (Jul/03/2015 )

Hello,

I would like to know will it be valid to use housekeeping genes which have a Cq value of 22 and GOI value of 28-29? Is the difference too much?

Also, wanted to know is 1 RQ value = 1 fold change OR 0.5 RQ = 1 fold change?

Lastly, has someone used this website http://bit.ly/1C785sV to know about the best HKG for their studies? It involves all the 4 methods i.e. Genorm, dct method, Bestkeeper and Normfinder?

-Mad Researcher-

Here's what my highly erudite friend has to say on the matter:

Hello

For what I understand of Q-PCR :

For comparing Cq values, you need primers that amplify your targets with the same efficiency. For that, do one standart curve for each couple of primers with serial dilution of your target. If you have good primers, the slope of each standart curve should be around -3,3. Comparing values of 22 and 28-29 is okay so long the Cq values are within the curve. In this case, the target with a Cq of 28 will be in 2^5=32 times less amount than the target with a Cq of 22 in your sample.

The website in the link no longer exists, I think.

Hope it helps

-gvbdxz-

gvbdxz on Tue Jul 7 11:08:17 2015 said:

Here's what my highly erudite friend has to say on the matter:

Hello

For what I understand of Q-PCR :

For comparing Cq values, you need primers that amplify your targets with the same efficiency. For that, do one standart curve for each couple of primers with serial dilution of your target. If you have good primers, the slope of each standart curve should be around -3,3. Comparing values of 22 and 28-29 is okay so long the Cq values are within the curve. In this case, the target with a Cq of 28 will be in 2^5=32 times less amount than the target with a Cq of 22 in your sample.

The website in the link no longer exists, I think.

Hope it helps

Thanks. I did a standard curve and all my primers have more or less the same efficiency.

For some reason the website is down since Saturday :( The website is known as ref-finder. I will let you know once the website is up and running.

-Mad Researcher-

Yay ok ! The website's probably taken a break to go out and get some fresh air. Can't blame it for wanting some

-gvbdxz-

gvbdxz on Tue Jul 7 12:15:18 2015 said:

Yay ok ! The website's probably taken a break to go out and get some fresh air. Can't blame it for wanting some

It was a nice website with all the algorithms. Hope it get backs soon. Really need it !!

Do you happen to know "is 1 RQ value = 1 fold?"

-Mad Researcher-