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
- - - - -

Can somebody explain to me what "spiking" means in RT-PCR and why do you

RT-PCR Spiking Positive control

Best Answer bob1, 19 November 2013 - 12:29 AM

You are correct - equivalent to a spiked control.  Basically you would add some DNA that you know will provide some signal (e.g. a plasmid or some synthetic DNA) to a reaction, and see if the reaction will amplify.

Go to the full post


  • Please log in to reply
3 replies to this topic

#1 That_Lab_Guy

That_Lab_Guy

    member

  • Active Members
  • Pip
  • 12 posts
1
Neutral

Posted 18 November 2013 - 11:48 PM

So I did a real time PCR lately to investigate the CT/copy number value of cDNA after doing a reverse-transcriptase PCR of pLVX-myc mRNA extracted from about 250 ng of MSC-derived exosomes.  The results were not exactly according to my expectations and I wanted to find out if there's anything in the exosome sample that is inhibiting the real-time PCR.

 

My boss has suggested a spiking when doing the real-time PCR.  I do not understand the principle and concept of doing this.  Can someone kindly explain to me clearly what is it and why is it done for certain PCR reactions?

 

If I am not mistaken, "spiking" here is equivalent to adding a "spike" control.

 

Thank you!



#2 bob1

bob1

    Thelymitra pulchella

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 5,713 posts
398
Excellent

Posted 19 November 2013 - 12:29 AM   Best Answer

You are correct - equivalent to a spiked control.  Basically you would add some DNA that you know will provide some signal (e.g. a plasmid or some synthetic DNA) to a reaction, and see if the reaction will amplify.



#3 That_Lab_Guy

That_Lab_Guy

    member

  • Active Members
  • Pip
  • 12 posts
1
Neutral

Posted 29 December 2013 - 08:54 PM

So is it correct to say that the purpose of a spike control is to show that the PCR machine is working fine?  That is to say that if I use a DNA sample that I know that should give a signal and that it does so accordingly, means there is nothing wrong with the machine?  So we can rule out the possibility of a technical fault when it comes to the PCR results.  

 

It is similar to a positive control, but different as this is adding a known DNA sample to an unknown sample.  Whereas a positive control is just adding the known DNA sample itself.


Edited by That_Lab_Guy, 29 December 2013 - 08:57 PM.


#4 mdfenko

mdfenko

    an elder

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 2,782 posts
132
Excellent

Posted 30 December 2013 - 04:49 AM

a spike is also used to normalize reactions to one another (for comparison) when you use a known amount of spike.


Edited by mdfenko, 30 December 2013 - 04:50 AM.

talent does what it can
genius does what it must
i do what i get paid to do





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

©1999-2013 Protocol Online, All rights reserved.