As Halloween approaches, I wanted to offer some juicy advice that will be of interest to the vampires, as well as the scientists, amongst our readership. I want to talk about blood [cue: thunder, lightning and evil laughter].

Whether you consider it a tasty treat, or a data-rich sample, blood, human or animal, is different from anything else you’ll work with. It is a complicated matrix of cells, plasma, and protein. Human erythrocytes number around 5 x 10⁹ cells per ml of blood, but because they do not have a nucleus, they contain no DNA (avian erythrocytes are nucleated and do indeed contain DNA, hence the need to start with 10 fold less blood for DNA extractions). And hemoglobin levels average around 150 mg/ml of blood. This high level of protein is a major issue in DNA and RNA contamination and PCR inhibition. The combination of cellular debris and protein make this sample as heavenly as candy corn for blood-sucking vampires, but not so for molecular biologists.

Anticoagulants:

One key point about working with blood is that the sample should always be collected in anticoagulant to prevent clotting. Isolation of DNA from clotted blood is not efficient and most of the cells will be lost in the clot.  Proteinase K digest of the blood clots will not work to release the DNA. The best you can do is remove the clot and start with any remaining liquid sample left in the tube.

The best anticoagulants to use are EDTA and Citrate. These do not interfere with downstream genetic analysis. Heparin is another anticoagulant sometimes used but is not advisable.  Heparin is highly negatively charged and will co-extract with the DNA inhibiting PCR (1).

Note to vampires: thanks to the potent anticoagulant, Draculin, contained in your saliva, you don’t need to worry about coagulation.

DNA from Blood

Getting DNA from blood is a lot easier than getting RNA. Blood can be fresh or frozen since DNA levels are much higher than RNA and DNase is much more labile than RNase.  However, repeated freeze thawing of blood should be avoided or DNA integrity will decrease with each new thaw.

The best way to store blood if you can’t extract DNA right away is at 4^oC for the short term (up to 2 weeks should be ok) and -20^oC or -80^oC for long term storage. Use -80^oC for archival storage.

The easiest method for isolating DNA from blood is a simple blood spin kit such as MO BIO’s UltraClean Blood Spin DNA Kit. With this method, 200 ul of blood is digested with proteinase K and a highly denaturing buffer for only 10 minutes. The DNA is next purified over a silica membrane and typical yields are around 4-6 ug per 200 ul.  If you need to start with more than 200 ul, then another fast and easy method is the non-spin kits that perform a basic lysis and isopropanol precipitation of the DNA. MO BIO offers these ready-to-go methods in a UltraClean Blood DNA Isolation Kit (300 ul blood) size, and then in kits that can process up to 1,000 ml of blood in any size volumes you wish. 

Blood DNA is, of course, considered a delicacy by vampires, so these kits are perfect for preparing your next dinner party. Blood DNA can be spiced with almond essence, crushed chili or oyster extract, depending on your tastes. But do NOT use garlic.

RNA from Blood

For RNA extraction from blood, your choices are much more limited. As every vampire knows, blood must be fresh to get the best result. RNases are tough scavengers and even frozen they will have activity. A freeze/thaw cycle only makes it easier for them to have access to their prey. Plus, lysis of WBCs during the thaw will mean loss of the RNA during the red blood cell (RBC) lysis step. To get the best quality RNA from blood, starting working on it immediately. We have stored blood at 4^oC for up to an hour before processing and obtained good quality RNA.

The first step in most RNA from blood extraction protocols is RBC lysis using a hypotonic lysis buffer. The result is a nice white blood cell pellet (WBC) that is easy to extract RNA from. If you can’t finish the RNA prep in the same day, your best bet is to get to this step and lyse the WBC pellet in a guanidine lysis buffer containing beta-mercaptoethanol (such as the one provided in the BiOstic Blood RNA Isolation Kit- details below) and store the pellets at -20^oC  or -80^oC.  Once you are ready to extract, warm up the sample completely to dissolve the salts and then proceed with the ethanol addition step. Always add the ethanol, (or red wine for vampires), fresh.

The BiOstic Blood RNA Isolation Kit

When we developed the BiOstic Blood RNA Isolation Kit we kept in mind the steps where the most damage can occur to the RNA and reduced them.  Every minute the cell pellet is not stabilized in the lysis buffer is time the RNA may be suffering from the RNA leeches. To combat this, we reduced the time and number of RBC lysis steps. This results in higher recovery of total WBCs and higher integrity RNA from the cells. We find that yields average between 0.10 µg and 0.5 µg/ 100 µl of human blood.

What about blood stabilized in PAXgene Blood RNA Tubes?

For processing blood that comes from another location, many labs are using the PAXgene Blood RNA Tube for collection and storage of samples. This reagent allows for collection of 2.5 ml of blood per tube and the entire amount is processed on a single spin column. Now that you know how rich in protein blood is, you can see what a tricky sample this would be.

Because the PAXgene collected sample contains a high level of WBCs, RBCs, and hemoglobin, specialized protocols are used for extracting RNA from this sample. For a normal person, 2.5 ml of blood contains around 60-80 ug of genomic DNA and between 4-12 ug of RNA. This is a lot of nucleic acids for a small spin column.

To extract RNA from PAXgene Blood RNA Tube samples, MO BIO Labs developed their own protocol in the BiOstic Stabilized Blood RNA Isolation Kit for homogenizing and releasing high levels of RNA from this very viscous pellet.  We found that by using a unique DNA shearing column containing our silica shearing beads, we could release more RNA from the debris compared to the Qiagen version of this kit.

PAXgene Blood RNA Tubes have also proven popular amongst vampire moms, since they can hold just enough tasty blood to keep the kids satisfied when you’re out and about. Why not give them a try?

Finally…

Whether you work with DNA, RNA, or just drink the stuff straight, working with blood requires care. Fortunately, there are many great commercial products available that make your life easier.

Don’t let a little blood scare you away from getting the results you need.  Have any questions? Looking for more tricks or have a problem? Leave us a comment and let us know how we can help.

Happy Halloween from MO BIO Labs!

[cue: thunder, lighting and evil laughter, again]

References:

1. Yokota M, Tatsumi N, Nathalang O, Yamada T, Tsuda I. (1999). “Effects of Heparin on Polymerase Chain Reaction for Blood White Cells”. J. Clin. Lab. Anal. 13: 133–140.

~ Suzanne (follow me on twitter)

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