I have been using HPLC purified primers for real-time PCR because it was recommended by the supplier. Has anyone tried without purification or Bio RP purified primers? Is HPLC purification essential for real-time PCR primers? Please suggest if any one has any idea or experience.
You have to look at the history of oligo synthesis in order to understand why commercial suppliers started suggesting HPLC grade oligos. Usually, the efficiency of oligo synthesis is never 100%. You can have a small fraction of "incomplete" oligos in the mix. For e.g., if you ordered a 24 base oligo, you can have a very small proportion of oligos with 22 or 23 bases. HPLC allows one to obtain pure 24 base oligos. However HPLC purification compromises oligo yield and costs more with no substantial improvement in PCR efficiency. HPLC purification need to be done only in very special instances; not for qPCR. With improvements in oligo chemistry and equipments, inefficiency of synthesis is not a major concern now a days. I presume that it is 98.5% or above and therefore good enough for PCR applications.
I have used real-time PCR primers usually after simple column-based purification, without HPLC. However, the PCR performance depends on annealing temperature, primer length etc. As long as you have standard condition, there is no HPLC needed, I guess
I would say you don't need to HPLC purify your primers. I have used primer provided by IDT in the most basic presentation (standard desalting) primers for real time without any problem. Used Syber Green as dye.
Our experience is the same as Martiniano's. We use IDT primers prepared with standard desalting and get good real time PCR results with Sybr green. Any problems with primer pairs are most likely due to the sequences of the primers--not purification. I have also used non-HPLC purified oligonucleotides as amplification primers in TaqMan assays.
We also use only standard desalting as primer purification and Sybr green for our real time PCR with excellent results.
I have used not hplc rt-pcr primers , ı purified primers only desalted method and using pcr.
I have used standard PCR grade primers for qPCR, do my mixing on the bench without a PCR hood and use standard sterile tips and not filter tips. I have obtained good results. I use the Illumina Eco Real Time PCR system, perhaps because the system software is robust?
I have used standard PCR primer for qPCR, it has given good results
Also, I use Kapa Biosystems Sybr Fast Mastermix for all my reactions
We have regularly used standard primers from ABI and no need to purify through HPLC.
No need for HPLC purification. Primer design is the most important aspect in terms of base composition, length etc to ensure your reaction efficiencies fall within the acceptable range.
Hello Satheesha, I m from CIFA in ur old lab, I also suggest you to use standard desalted primers for REAL TIME PCR without any problem . However parameters for design the primers are important.
Thanks to all, Yes me too was thinking the same as all you have suggested. But the supplier had recommended to go for HPLC purification (it costed $25-30 ! good business) but they did not give any proper reason for my question 'why'. So thought to put that question here. Thanks all for confirming my decision.
it's a very difficult question for suppliers : They can only answer "because they are better than unpurified ones". In that case, you would ask "does this mean that the oligo you were selling up to now are not optimal, in spite of you always saying they are great ?".
Besides that, there may be some interest in purification. For instance, impurities can impair oligonucleotide preservation.
You have to look at the history of oligo synthesis in order to understand why commercial suppliers started suggesting HPLC grade oligos. Usually, the efficiency of oligo synthesis is never 100%. You can have a small fraction of "incomplete" oligos in the mix. For e.g., if you ordered a 24 base oligo, you can have a very small proportion of oligos with 22 or 23 bases. HPLC allows one to obtain pure 24 base oligos. However HPLC purification compromises oligo yield and costs more with no substantial improvement in PCR efficiency. HPLC purification need to be done only in very special instances; not for qPCR. With improvements in oligo chemistry and equipments, inefficiency of synthesis is not a major concern now a days. I presume that it is 98.5% or above and therefore good enough for PCR applications.
Using standard primers from ABI, there is no need to purify through HPLC.
The coupling efficiency per base for traditional oligo synthesis approaches such as beta-cyanoethyl phorphoramidite chemistry is approximately 99%. A quick way to calculate the total number of species with full length in your oligo mixture is to raise the percentile by the total number of bases in your primer. Suppose your primer is 18 bases in length you will calculate your purity as 0.99^18 = approx 83%. The only thing you get with HPLC purification is a guaranteed percent purity, which is greater or equal to 85% for most commercial suppliers. Unless you are using labeled probes, which need to be purified to ensure removal of free uncoupled dyes, you are not really getting much in difference in purity between your de-salted and HPLC purificed primers.
Thanks to all once again for such a detailed information. I used to order a Korean company Bioneer, which provide BioRP purified oligos without additional cost. They had recommended to go for HPLC purification, But recently found that BioRP purification is better than simple desalted primer and is near HPLC purified primer, in quality. That made me to put the question here. Now I am working with the BioRP purified primers and is giving really as same as HPLC purified primer. With no difference in CT values or in any noisy background reactions.
I want to order primer sets of RT-qPCR for miRNA detection based on the P K Busk et al paper ( without probe ) .
Is the DNA Reverse Phase Column purification method is proper for this aim ?
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