Hello Rais
as alluded to above its all about primer efficiency and specificity which comes down to designing good primers and ensuring optimal q(RT) PCR conditions; in particular matching the annealing temp of primers to their melting temp to maximise efficiency and specificity
1. The single most effective thing you can do is pre screen primers by agarose gel electrophoresis and proceed to actual qPCR with the best set. Specifically evaluate primers at Tm - 2 C as annealing temp to verify specificity and if that yields multiple non specific bands try an annealing gradient from Tm+1C to Tm-5C in 1C intervals. Pick the annealing temp that yields the strongest band but specifically. Then use this same annealing temp in your actual qPCR
2. As already mentioned, perform a standard curve by diluting your cDNA from 1:1 to 1:10,000 in log 10 increments for high copy number genes and a 1:2 serial dilution of your cDNA from 1:1 to 1:128 for low copy number gene. Pick the lowest serial dilution that yields consistent triplicates with reference to Ct cycle value; and demonstrates a consistent act value in the ambient range of 15 to 25 cycles
3. In addition with this dilution of cDNA and verified annealing temp make sure your melt curves mirrors evidence from agarose gel I. E one specific melt peak recapitulating one string band on an agarose gel
4. For highest efficiency also ensure your amplicon is preferable about 100bp and certainly no more than 300bp: in general this is conducive to amplification at or above 85% efficiency; a pre requisite for accurate qPCR
5. In terms of target placement bias your amplicon towards the 3' end of the transcript as reverse transcription proceeds from the 3' to the 5' end of your RNA so the latter part of the transcript is most efficiently and accurately represented. This is especially true when performing RT with oligo dT; you are less restricted with random hexamers. Specifically if making cDNA with oligo dT alone try and locate your 100-300bp target amplicon 1-2kb from your poly A tail
Finally when making primers use many of the tools available on line like IDT oligo analyser to design by eye and screen for things like hair pin loops or the possibility of duplex formation between forward and reverse primers which can lead to primer diner formation and drop your reaction efficiency below 85% which you require for accurate qPCR; better still utilise one of a myriad of primer design tools on line like IDT primer quest; primer 3; or primer blast. These will automatically allow you to submit a region of interest and design good primers with compatible Tms and screened for above complications. Primer Blast in particular is especially useful as it allows you to easily make exon spanning primers from Ref sequences in Genbank. These will only target cDNA and not prime parent genomic DNA
Good luck !
What sort of real time PCR? If you mean rt-qPCR, the two main points are:
1) Optimise your primers to identify their effiiciency (http://www.sigmaaldrich.com/technical-documents/protocols/biology/primer-concentration-optimization.html)
2) Make sure your PCR results show one peak at dissociation curve. If not, there are ways to remove additional peaks, e.g. decrease primer concentration to prevent self complementarity, but I would just buy a new pair of primers.
Hello Rais
as alluded to above its all about primer efficiency and specificity which comes down to designing good primers and ensuring optimal q(RT) PCR conditions; in particular matching the annealing temp of primers to their melting temp to maximise efficiency and specificity
1. The single most effective thing you can do is pre screen primers by agarose gel electrophoresis and proceed to actual qPCR with the best set. Specifically evaluate primers at Tm - 2 C as annealing temp to verify specificity and if that yields multiple non specific bands try an annealing gradient from Tm+1C to Tm-5C in 1C intervals. Pick the annealing temp that yields the strongest band but specifically. Then use this same annealing temp in your actual qPCR
2. As already mentioned, perform a standard curve by diluting your cDNA from 1:1 to 1:10,000 in log 10 increments for high copy number genes and a 1:2 serial dilution of your cDNA from 1:1 to 1:128 for low copy number gene. Pick the lowest serial dilution that yields consistent triplicates with reference to Ct cycle value; and demonstrates a consistent act value in the ambient range of 15 to 25 cycles
3. In addition with this dilution of cDNA and verified annealing temp make sure your melt curves mirrors evidence from agarose gel I. E one specific melt peak recapitulating one string band on an agarose gel
4. For highest efficiency also ensure your amplicon is preferable about 100bp and certainly no more than 300bp: in general this is conducive to amplification at or above 85% efficiency; a pre requisite for accurate qPCR
5. In terms of target placement bias your amplicon towards the 3' end of the transcript as reverse transcription proceeds from the 3' to the 5' end of your RNA so the latter part of the transcript is most efficiently and accurately represented. This is especially true when performing RT with oligo dT; you are less restricted with random hexamers. Specifically if making cDNA with oligo dT alone try and locate your 100-300bp target amplicon 1-2kb from your poly A tail
Finally when making primers use many of the tools available on line like IDT oligo analyser to design by eye and screen for things like hair pin loops or the possibility of duplex formation between forward and reverse primers which can lead to primer diner formation and drop your reaction efficiency below 85% which you require for accurate qPCR; better still utilise one of a myriad of primer design tools on line like IDT primer quest; primer 3; or primer blast. These will automatically allow you to submit a region of interest and design good primers with compatible Tms and screened for above complications. Primer Blast in particular is especially useful as it allows you to easily make exon spanning primers from Ref sequences in Genbank. These will only target cDNA and not prime parent genomic DNA
Good luck !
Dear Rais
For optimization of RT-PCR, run the serial dilution of template (DNA/RNA) and generate standard curve. The standard curve is constructed by plotting the log of the starting quantity of template against the Ct values obtained at amplication of each dilution. The equation of the linear regression line, along with Pearsons correlation coefficient (r) or conefficient of determination (R2), can be used to evaluate whether ur qPCR assay is optimized.
All the best!
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