I am starting work with qPCR of human IFN (SYBR green). I need evaluate the level of expression of human IFN alpha and IFN gamma and I have some questions.
- can I use forward primer for cDNA?
- does anyone have a reference articles about the sequence of primers for detection of human IFN alpha and IFN gamma?
I have been developing human and murine IFN qPCR assays for several years and have had variable success, even with primers that are highly-specific, in-silico. Others have also reported stochastic expression of IFNB1.
Most interferon genes are intronless, making the mockRT control reaction absolutely necessary to be confident that the signal is specific to cDNA synthesis. Regardless of the priming strategy, you want to see Ct/Cq values in the plusRT reactions at least 3 Ct/Cq units lower than the mockRT. Occasionally, I have found a mockRT reaction gernerate a lower Ct/Cq value than the plusRT.
Since the genome is dynamic and database annotation is frequently revised and updated, you should BLAST the primer sequences before using them in your experiments to re-confirm specificity and potential cross-reactivity.
It is a challenge, if not impossible, to design primers specific to the IFNa genes. There are ~ 20 IFNa genes in the locus, Copy number, SNPs and rearrangement will likely vary from donor to donor and over time as cells divide and/or experience stresses.
In theory, using a forward (sense) primer to synthesize cDNA should not work. If you find that you are able to generate a specific signal using a gene specific sense primer to prime the RT reaction, the cDNA was likely primed via a self-priming mechanism. Try another control RT reaction where no exogenous primers are added to the cDNA synthesis reaction. For some targets, I have found that the addition of RT primers (random hexamers) produces an 8 Ct/Cq unit left shift when compared to the no exogenous primer control reaction. The noExoPrimer control generated a Ct/Cq value of 25, while the mockRT reaction showed no amplification.
Proper assay validation is very important. Amplification efficiency is likely to vary between cells and with treatments. Compare a few cDNA synthesis kits using different reverse transcriptases. Since IFNs are innate immune system molecules and part of the anti-viral response, there are likely to be differences between various RT enzymes' sensitivity to endogenous inhibitory molecules or gene-specific RNA modifications. Please follow up with any more questions.
Good Luck,
Tom
Hi Ilya,
I couldn't get your first Q. If you worry about gDNA contamination in your assay, you just need to use minusRT control on all your samples.
For second Q. : Some variants of IFNa and IFNg qPCR probes you could find in Supplementary2 info in: http://www.nature.com/icb/journal/v90/n8/suppinfo/icb2011109s1.html
It is not a SYBR green one but should work with SYBR too.
Here is IFNa probes used with SYBR: http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0024252
I have been developing human and murine IFN qPCR assays for several years and have had variable success, even with primers that are highly-specific, in-silico. Others have also reported stochastic expression of IFNB1.
Most interferon genes are intronless, making the mockRT control reaction absolutely necessary to be confident that the signal is specific to cDNA synthesis. Regardless of the priming strategy, you want to see Ct/Cq values in the plusRT reactions at least 3 Ct/Cq units lower than the mockRT. Occasionally, I have found a mockRT reaction gernerate a lower Ct/Cq value than the plusRT.
Since the genome is dynamic and database annotation is frequently revised and updated, you should BLAST the primer sequences before using them in your experiments to re-confirm specificity and potential cross-reactivity.
It is a challenge, if not impossible, to design primers specific to the IFNa genes. There are ~ 20 IFNa genes in the locus, Copy number, SNPs and rearrangement will likely vary from donor to donor and over time as cells divide and/or experience stresses.
In theory, using a forward (sense) primer to synthesize cDNA should not work. If you find that you are able to generate a specific signal using a gene specific sense primer to prime the RT reaction, the cDNA was likely primed via a self-priming mechanism. Try another control RT reaction where no exogenous primers are added to the cDNA synthesis reaction. For some targets, I have found that the addition of RT primers (random hexamers) produces an 8 Ct/Cq unit left shift when compared to the no exogenous primer control reaction. The noExoPrimer control generated a Ct/Cq value of 25, while the mockRT reaction showed no amplification.
Proper assay validation is very important. Amplification efficiency is likely to vary between cells and with treatments. Compare a few cDNA synthesis kits using different reverse transcriptases. Since IFNs are innate immune system molecules and part of the anti-viral response, there are likely to be differences between various RT enzymes' sensitivity to endogenous inhibitory molecules or gene-specific RNA modifications. Please follow up with any more questions.
Good Luck,
Tom
Dmitry, thank you for references.
Thomas thanks a lot for the deployed answer. If I right understand, you advise me to use the random primer to synthesize cDNA?
Random hexamers are a good place to start. If your target region is at the 3' end, oligo dT alone or in combination with random hexamers may improve your results. A gene-specific priming strategy may improve specificity of the IFNa assay. i have not had a problem with IFNG amplification.
Sequencing the qRT-PCR products revealed that IFNa primer sets amplify other IFNa amplicons. Perhaps high homology between the variants leads to strand exchange during PCR extension. The gene specific RT priming strategy may help.
These IFNG primers have worked well for me, however should be validated in your system
hsIFNG-f GTA TTG CTT TGC GTT GGA CA
hsIFNG-r GAG TGT GGA GAC CAT CAA GGA
hsIFNG-r TTG GCT TTT CAG CTC TGC ATC GTT
hsIFNG-r TCC GCT ACA TCT GAA TGA CCT GCA T
hsIFNG-f GAA ACG AGA TGA CTT CGA AAA GCT GAC
hsIFNG-r CTG CTG GCG ACA GTT CAG CCA T
Good Luck!
Thomas thanks a lot again. I hope it will be helpful for my further work
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