I want to study the expression pattern of my genes of interest. Since i am operating RT PCR for the first time i am not aware of some of the things related to RT PCR. I want to know the difference between two step reaction and three step reaction.What is more appropriate? Let me tell you that we have RT PCR of corbett rotor gene 6000. Is it necessary to obtain melting curve and what is the purpose of melting curve analysis? My control gene is BETA ACTIN. and i am using SYBR Green containing master mix.
Purpose of melting curve analysis is to check that your primers are giving a specific PCR product and there is no non-specific amplification. More than one peak shows nonspecific amplification.
But before you rely on the melting curve do (once, just for establishing the PCR) a gel as well. Only then you can say that/if the melting peak you see corresponds to the specific product. (actually, only sequencing would assure this, but getting a product of the correct length is good enough in >99% of the cases). After you have shown this correspondance once for your setup (primers/reaction/sample/instrument) you can subsequently rely on the melting curves alone.
Note that very different PCR procucts can have quite similar apparent melting temperatures!
Melting curves almost always distinguish well between some "templeted" PCR product (what is hopefully the wanted, specific product) and "primer-dimers" that often have a considerably lower Tm. Such reactions will giive you a Ct value, but quick look at the melting curve tells you that this Ct value is "erroneous" (= does not correspond to the amount of target sequence).
Thank you all for your answers...I also want to know that how to interpret the results obtained after RT PCR?
This will help regarding any questions you have using SYBR green. Real-time PCR by Tevfik Dorak. I just recently started using SYBR green and i found the book helpful in terms of interpreting results as well. Good luck .
given that nonspecific and primer-dimer products can be formed during PCR reaction ,a melting curve analysis is highly recommended to check the specificity of the amplified fragments.
@Tega opuoro where can i get the book for interpetation of RT-qPCR results....especially on melting curve,, excel data sheets and et-cetra....@Nadine a .Gund where can i get the paper on RT-qPCR interpretation of results
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