Jump to content

  • Log in with Facebook Log in with Twitter Log in with Windows Live Log In with Google      Sign In   
  • Create Account

Submit your paper to J Biol Methods today!
Photo
- - - - -

always run internal control for different genes on the differnet plates?


  • Please log in to reply
8 replies to this topic

#1 catheriney

catheriney

    member

  • Active Members
  • Pip
  • 5 posts
0
Neutral

Posted 27 January 2010 - 12:42 PM

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.

#2 fishdoc

fishdoc

    Veteran

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 272 posts
2
Neutral

Posted 27 January 2010 - 01:01 PM

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.

#3 nightingale

nightingale

    Veteran

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 191 posts
7
Neutral

Posted 27 January 2010 - 01:01 PM

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.
" The more you learn, the more you realize how little you know ... "

#4 catheriney

catheriney

    member

  • Active Members
  • Pip
  • 5 posts
0
Neutral

Posted 27 January 2010 - 01:20 PM

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.

#5 fishdoc

fishdoc

    Veteran

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 272 posts
2
Neutral

Posted 27 January 2010 - 01:48 PM

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.

#6 nightingale

nightingale

    Veteran

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 191 posts
7
Neutral

Posted 28 January 2010 - 12:12 PM

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

hope some one will help ..

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

" The more you learn, the more you realize how little you know ... "

#7 stylothecancer

stylothecancer

    Enthusiast

  • Active Members
  • PipPipPipPipPip
  • 42 posts
0
Neutral

Posted 28 January 2010 - 01:07 PM

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.

=)

#8 AussieUSA

AussieUSA

    Enthusiast

  • Active Members
  • PipPipPipPipPip
  • 32 posts
0
Neutral

Posted 29 January 2010 - 02:35 PM

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.

#9 catheriney

catheriney

    member

  • Active Members
  • Pip
  • 5 posts
0
Neutral

Posted 30 January 2010 - 08:45 AM

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




Home - About - Terms of Service - Privacy - Contact Us

©1999-2013 Protocol Online, All rights reserved.