Heparin is a highly charged biological molecule -polymeric glucosaminoglycan. It prevents coagulation of blood but can be co-purified with the nucleic acids depending upon the preparation method used. It is a well known inhibitor of PCR and preferably should be avoided if collecting blood for PCR analysis. It can be digested with heparinise, but not really worth the effort . EDTA and Citrate are both used as anti-coagulants. The use of different coagulants depends upon the downstream medical / biochemical analysis being undertaken - eg plasma enzyme levels etc. EDTA is a chelator and an inhibitor of PCR as it can chelate Mg2+ ions, but the extraction procedures and EtOH pptn steps usually result in the majority of EDTA being removed. It can be an issue if you are amplifying from whole unfractionated blood, but can be overcome with addition of extra MgCl2 - unlikely for real-time PCR applications.
Citrate is the most benign and the best choice.
If you think you have an issue with sample PCR inhibition, spike a known PCR positive sample (eg plasmid) with a your extracted target and see if you get / lose amplification.
Ian
Yes, I know the basics, but I meant details? Advantages/disadvantages/experiences/ troubleshooting when doing real time PCR.
Heparin is a highly charged biological molecule -polymeric glucosaminoglycan. It prevents coagulation of blood but can be co-purified with the nucleic acids depending upon the preparation method used. It is a well known inhibitor of PCR and preferably should be avoided if collecting blood for PCR analysis. It can be digested with heparinise, but not really worth the effort . EDTA and Citrate are both used as anti-coagulants. The use of different coagulants depends upon the downstream medical / biochemical analysis being undertaken - eg plasma enzyme levels etc. EDTA is a chelator and an inhibitor of PCR as it can chelate Mg2+ ions, but the extraction procedures and EtOH pptn steps usually result in the majority of EDTA being removed. It can be an issue if you are amplifying from whole unfractionated blood, but can be overcome with addition of extra MgCl2 - unlikely for real-time PCR applications.
Citrate is the most benign and the best choice.
If you think you have an issue with sample PCR inhibition, spike a known PCR positive sample (eg plasmid) with a your extracted target and see if you get / lose amplification.
Ian
Dear Milos
The blood collecting tubes are covered with a color-coded plastic cap. They often include additives that mix with the blood when collected, and the colour of the tube's plastic cap indicates which additives that tube contains. The tubes may contain additional substances that preserve the blood for processing in the medical laboratory. Using the wrong tube may therefore make the blood sample unusable. These substances are typically a thin film coating that is applied using anultrasonic nozzle. The substances may include anticoagulants (EDTA, sodium citrate, heparin) or a gel with intermediate density between blood cellsand serum.
Types of Containers containing anticoagulants & their Importance
1. Green - Contains suheparin or lithium heparin used for plasma determinations in clinical chemistry (e.g. urea and electrolyte determination).
2. Light Green or Green/Gray 'Tiger': For plasma determinations.
3. Purple or lavender - contains EDTA (the potassium salt, or K2EDTA). This is a strong anticoagulant and these tubes are usually used for complete blood counts(CBC) and blood films. Lavender top tubes are generally used when whole blood is needed for analysis. Can also be used for some blood bank procedures such as blood type and screen, but other blood bank procedures, such as crossmatches must be in a pink tube in most facilities.
4. Grey - These tubes contain fluoride and oxalate. Fluoride prevents enzymes in the blood from working, by preventing the stage called glycolysis so a substrate such as glucose will not be gradually used up during storage. Oxalate is an anticoagulant.
5. Light blue - Contain a measured amount of citrate. Citrate is a reversible anticoagulant, and these tubes are used for coagulation assays. Because the liquid citrate dilutes the blood, it is important the tube is full so the dilution is properly accounted for.
6. Dark Blue - Contains sodium heparin, an anticoagulant. Also can contain EDTA as an additive or have no additive. These tubes are used for trace metal analysis.
7. Pink - Similar to purple tubes (both contain EDTA) these are used for blood banking.
8. Black - Used for ESR also known as Erythrocyte Sedimentation Rate or "sed rate." Goes to hematology department.
9. Red (glass)- Contains no additives. Tests for antibodies and drugs often require these.
10. Light yellow - Contains sodium polyanethol sulfonate (SPS). Used for blood culture specimens or acid-citrate-dextrose (ACD), used for blood bank studies, HLA phenotyping, and parental testing.
11. Tan (glass or plastic) - Contains either sodium heparin (glass) or K2EDTA (plastic). Used for lead determinations. These tubes are certified to contain no lead.
Best Regards
First tnx for the answers :-). I have been isolating lymphocytes from whole blood using EDTA coated tubes with adding Ficoll and etc. Recently my college gave mi some 15 ml BD vacutainer CPT tubes but the yield was very disappointing> After I optimize my isolation prep with this CPT tubes am I planing on doing real time PCR.
Hi Milos
I am currently investigating different methods for isolating lymphocytes from whole blood as well. Firstly, what CPT tubes are you using? We have only been able to find 8mL CPT tubes with sodium citrate so far but 15mL would be much more suited to our purpose. Do the CPT tubes you are using come with sodium citrate or heparin coated?
Also, what was the yield from CPT tubes vs standard tubes with Ficoll added?
Thanks very much
Right now. I also have the same issue and if you can suggest me something from your experiments. Which Vacutainer did I use and why and any suggestions related to ideal storage, temperature of the blood tubes to get the good quality of RNA and DNA ?
Do you think that capillaries with heparin would inhibit PCR? Or that would not be enough?
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