We already purified DNA from human PBMCs that were originally collected in sodium heparin tubes. Since we know heparin can affect qPCR reactions (which we would like to use DNA for SNP analysis), is there a good protocol to try to remove the heparin or is there an enzyme that is less affected by heparin? Or do we just have to collect blood again in EDTA?
I had to use DNA that had been extracted with phenol-chloroform ... y repurified it using columns... it worked quite well in qPCR... good luck
In our hands heparin inhibited PCR in case when we have extracted DNA/RNA from whole blood. Since the PBMC washed out of rest assume that inhibition effect is minimal.
Hi Deanna, We have used PBMC collected in heparin tubes for RTqPCR. and we did not had any problem with the PCR reaction. I believe the gradient centrifugation during, RBC lysis, and multiple PBS washes cell isolation and finally the RNA extration should almost eliminate the heparin; and it should not interfere with any PCR reaction. you may try washing the cells with 10 ml of PBS before lysis. I dont know what method you use for DNA extraction but, I think that when you precipiate DNA, and discard everythingelse it should get rid of heparin also. you may want to wash your DNA precipitate with 70% ethanol.
If it is just for genotyping purposes, you could also use AMpure XP beads to purify your DNA. It is highly efficient, quick, easy and inexpensive.
Purifying the samples with the Nexttec columns could help. They bind impurities such as proteins and your DNA just flows through. Nice and easy if you need high throughput solutions.
There are mutliway ways to get rid of low-molecular-weight contaminants from DNA. Your method shoud be reliable, cheap and fast, I quess. In our experience, simple precipitation of DNA with 2.5 volume of ethanol at room temperature, immediate and brief (3 mins)centrifugation in an epp. centrifugation of sufficient of remove most of non-proteinous compounds.YOu may wish to wash the precipate with 70% Ethanol before drying. If speedvac is applied for drying the whole procedure should not take you more than 15 mins. .
I use Kurabo blood DNA extraction kit with Fuji mini extractor, from blood taken on heparin, and works perfectly for PCR.
Instead of heparin ACD (acid citrate dextraose) can be used as an anti-coagulant, which does not inhibit PCR or any other cell culture techniques.
For qPCR genotyping you have to use minimal quantities of DNA, so I think you will have no interference of heparin. Try a number of reactions with long protocols (i.e 50 cycles) before going for expensive DNA repurification. IIf you really have problems amplificating samples, you may choose to use a column or better, precipitation based methods (i.e. salting out or some kit like Puregene form Qiagen or Masterpure from Epicentre, both with very good results in our hands). Be sure you keep a aliquot of your sample, since usually you loose quite a bit of DNA when repurifying it. Good luck!
You can try pretreating the DNA with Heparinase, an enzyme that breaks down heparin.
Sorry had not checked researchgate in awhile but thank you all for your responses. I do think the fact we have PBMC versus from whole blood this may help a bit to reduce heparin contamination but I think we will just reprecipitate to clean up a bit more since we have extra DNA. As Rocio mentioned since genotyping doesnt require a lot of DNA then we may be okay. For future I will just use EDTA tubes I think if for genotyping but for culturing cells I definitely prefer heparin in case EDTA inhibits immune cell stimulation so hopefully as mentioned all the washes will remove the heparin before actually running the qPCR.
You have to wash away the Heparin, before you put the leukocytes in the lysis buffer. Don't freeze heparinized bloodsamples. Heparinase treatment works sometimes. Heparin binds irreversible to DNA, so there is no method to remove it except Heparinase.
Martin is correct about Heparin binding directly to DNA. It is well known in the forensic DNA community that Heparin blood samples are notorious for inhibiting PCR. You cannot wash it off of the DNA. You have to change your isolation method to one which prevents it from binding in the first place. I do not know which kit is best, specifically for heparin (anyone?). However, if you do a manual isolation, I can tell you how to prevent heparin contamination in the DNA. Let us know how it goes!
Hello Deanna.
No worries. In cases like this I use Hemo Klen Taq (New Enbland BioLabs). The enzyme works perfectly in the presence of heparine.
Not for TaqMan style qPCR! A quote from NEB website on that product:
"Product Source - An E. coli strain that carries a mutant Taq DNA polymerase gene. The protein lacks the N-terminal 5´→ 3´ exonuclease domain and the gene has three internal point mutations." This exo is required for TaqMan activity.
For qPCR in general you have to use the same DNA isolation protocol for al samples. Otherwise you can not compare the results to a reference in which the DNA has been isolated in a different way. The presence of inhibitors can differ between isolation method.
I agree Martin. Only lost 2 samples as the other times we simultaneously collected in EDTA and Heparin so will just use EDTA PBMC DNA for genotyping. It seems like most people though use PBMC for stimulations isolated from heparinized blood and then isolate RNA for qRT-PCR for looking at inflammatory genes so the heparin must be mostly washed off for the PBMC stim for the qRT-PCR to work if it's such common practice.... Thanks!
We routinely prepare cDNA from mast cell tumours for qRT-PCR and as you would expect these are laden with heparin. But heparinase works fine for us.
Simply, try to aliquot your blood sample in two tubes one heparinised for PBMC stimulation and another on EDTA for qRT-PCR . good luck!
I would try Heparinase treatment. The following paper seems interesting:
Biotechniques. 2003 Dec;35(6):1140-2, 1144.
Heparinase treatment of RNA before quantitative real-time RT-PCR.
Johnson ML1, Navanukraw C, Grazul-Bilska AT, Reynolds LP, Redmer DA.
We are using heparinase which we add to standard isolated DNA from sodium heparine tubes with very good results according to the following protocol:
Order 50 units of Heparinase I from Sigma (product ID: H-2519). Directly to this bottle, add 11.2µl of 1M Tris pH 7.5, 2.2 µl of 1M CaCl2 and 2229µl of water. Divide this solution into 19µl aliquots and store at -20°C.
For use, simply thaw out a 19µl aliquot of the above solution, add 4µl of your DNA sample (assuming an average concentration of 400-600 ng/µl) and place at 37°C for 2 hours. Use 5µl of the treated DNA solution as your starting template in subsequent MLPA reactions.
A.C. Taylor (1997) Titration of heparinase for removal of the PCR-inhibitory effect of heparin in DNA samples Molecular Ecology 6;383-385.
Just thought I would update to say if you isolate PBMCs from heparinized blood and then purify nucleic acid we have NOT had inhibition of qPCR so as a few mentioned, must be a big problem only if isolate directly from blood.
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