8 replies to this topic

[catheriney]

Hi, I want to use qPCR to check the different gene expressions and I use 16S rRNA as the internal control to normalize my samples. Should I always run 16S rRNA on different plates when I check different genes for the same set of samples?
My rational is that since I have already run 16S RNA for different samples and they shouldn't change from plates to plates, so it is not necessary to run 16S on every single plate, am I right? Thank you.

[fishdoc]

catheriney, on Jan 27 2010, 02:42 PM, said:

Hi, I want to use qPCR to check the different gene expressions and I use 16S rRNA as the internal control to normalize my samples. Should I always run 16S rRNA on different plates when I check different genes for the same set of samples?
My rational is that since I have already run 16S RNA for different samples and they shouldn't change from plates to plates, so it is not necessary to run 16S on every single plate, am I right? Thank you.

It "shouldn't" change from plate to plate... but it could. If you don't run it, you won't know if it's the same or not. I would say run it. It allows you to be sure the plates are comparable in template by showing your endogenous isn't changing between plates.

[nightingale]

well,
here is what i thought :
what if u ended up with a sample showing no signal...
how will u know that it is really negative ??

i mean :
reading the IC will assure that there are no inhibitors in my samples,
which will avoid false negative results.

[catheriney]

thanks.
It is just I am using the standard curve method and if I run internal control every plate, half of my plate will be the 16S. In this case I can only check one gene per plate for only 12 samples which seems a big waste to me.

[fishdoc]

catheriney, on Jan 27 2010, 03:20 PM, said:

thanks.
It is just I am using the standard curve method and if I run internal control every plate, half of my plate will be the 16S. In this case I can only check one gene per plate for only 12 samples which seems a big waste to me.

I usually use delta delta Ct method, which is what I based my above response. I'm not familiar enough with the standard curve method to comment on the need for 16s on each plate.

[nightingale]

sorry catheriney ...
also, not familiar with your method ...

hope some one  will help ..

Edited by nightingale, 28 January 2010 - 12:13 PM.

[stylothecancer]

Hi catheriney,

I will still running the the internal control.  To me, i will be more confident with my data if I did so; actually I did so all the time.  That's why I prefer to run my real time PCR in 384-wells plate, rather than 96-wells plate.

=)

[AussieUSA]

Everytime you run a plate, there could be a slight difference in amplification based on your pipetting ability, the temperature of the room, you name it. Therefore you should always run the standard on every plate you amplify.

As Stylo states ... a 384-well plate could save you for space.

Have fun,

AussieUSA.

[catheriney]

thanks a lot!
i should get the 348-well plate then...