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Can somebody explain to me what "spiking" means in RT-PCR and why do you - (Nov/18/2013 )

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!

-That_Lab_Guy-

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.

-bob1-

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.

-That_Lab_Guy-

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

-mdfenko-