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