I am looking for some protocol which will help me to increase the intensity of PCR for Toll-like receptors 4.
A suspect that you talk about RT-PCR (analysing cDNA). And I further guess that "intensity of PCR" means product yield.
You can use sequence-specific primers in the RT.
You can perform a nested PCR (first PCR with "outer" primers, then use the PCR product as template for a second PCR using "inner" primers).
hi
1- you can increased 1 or 2 Cycles in PCR process ( 37 cycle ) .
2- you can used from 1 ul DMSO in PCR working solution ( 25 ul total PCR reaction volume ) With the aim of opening GC loops and increase the intensity of PCR band.
good luck
Thank you so much for your responses and I am sorry, I was not very specific.
So the problem is that curve for TLR4 is coming up in 35th cycle which is late. Probably the quantity of RNA is low or there could be some contamination, so I am looking for some protocol or tip, which will help me to make whole reaction more sensitive.
We can use more concentration of RNA but we would like to find another possibilities.
hi
As you said yourself, it's time to extract RNA observe sterile conditions and solve this problem using more specific primers. I think these articles can help you.
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3586846/
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1809579/
https://www.biomol.com/product_TLR4-Human-toll-like-receptor-4-Real-Time-PCR-Primer-Set.html?aRelated=VHPS-9314
good luck
So you do real-time qPCR. A Ct of 35 is obtained under ideal conditions (amplification efficiency = 2) and usual setups for abot a single molecule in the PCR. I assume you validated your PCR assay so you are sure the amplification efficiency is close to 2.0. Then your samples simply contain nearly no target molecules and the only way to get quantifiable amounts is to increase the number of target molecules in the PCRs.*
Try the RT with gene-specific primers. You can mix the primers for the different genes you want to quantify (target + reference genes). Use the one primer per gene that is complementary to the mRNA in a final concentration of 0.2-0.5 µM.
Make sure your samples are well purified (don't contain RT inhibitors) and use an RT enzyme with high yield (like sngineered MMLV).
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* regarding "to make whole reaction more sensitive": the PCR should actually be optimally sensitive. If there is a single molecule, you will get a positive reaction. A reaction cannot be more sensitive than that (it can only be less specific and giving positive results in cases where there is no molecule in the mix). Getting a lower Ct for the same amount of target molecules does not mean to increase the sensitivity. This may only have an impact on the signal-to-noise ratio and the limit of quantification (LoQ) - parameters you should know from the assay validation.
Ok, I understand. Thank you so much for such detailed answer. I am not very experienced with PCR and I will go through it with my supervisor, I am sure it will help us a lot! Thank you.
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