Two vampires walk into a bar and call for the bartender.
“I’ll have a glass of blood,” says one.
“I’ll have a glass of plasma,” says the other.
“Okay,” replies the bartender, “That’ll be one blood and one blood lite.”
(insert laughter here)
Yep, we are celebrating the scary season and launching our new PowerMag Blood DNA/RNA Isolation kit!
Blood: What’s in it?
Blood contains a mixture of plasma, red and white cells and platelets. It is a unique beast among sample types, because while the quantity of nucleic acids in blood is copious, this genetic gold mine bathes inside a complex soup of cellular debris and protein. These contaminants can interfere with downstream PCR and sequencing. It is the hemoglobin in particular, within the red cells, which causes major issues in DNA/RNA contamination and PCR inhibition.
Fortunately, dirty samples don’t scare us at here at MO BIO. After all, we know how to get clean nucleic acids from feces and soil, no problemo!
Blood Collection and Storage:
Blood should be collected into an anticoagulant coated tube. Otherwise, the blood will clot and bind up most of your DNA/RNA containing cells. Standard blood collection tubes typically contain EDTA or citrate to prevent this. Neither of these interferes with downstream genetic analysis. Heparin can also prevent clotting but because it tends to bind to DNA, we don’t recommend it.
If whole blood can’t be processed right away it’s okay to store it at 4oC for up to two weeks for DNA isolation or for an hour or two for RNA isolation. Any longer than this and you’ll need to take alternative action. If you only want DNA you can simply freeze the blood in small aliquots (250-500 μL) at -20oC or -80oC for long term storage.
For RNA extraction from blood, you’ll need to be more vigilant. RNases are tough enzymes and they can continue to have activity even while frozen. If the blood cannot be processed quickly then we recommend collecting the blood in an RNA stabilization buffer such as that contained in PAXgene™ Blood RNA Tubes. In these tubes, intracellular RNA will be stable for three days at 18 to 25°C or five days at 2 to 8°C.
Another option, if you can’t get to your RNA prep right away is to isolate, lyse and freeze the white cell pellet in an RNase inhibiting buffer. For example, the white pellet can be stored in the PowerMag® WBC Lysis solution contained in the PowerMag Blood DNA/RNA Isolation kit. (See below) Store the pellets at -20oC or -80oC. Once you are ready to extract, bring the sample to room temperature and proceed with the rest of the protocol.
In our PowerMag Blood DNA/RNA Isolation kit, the lysis of RBC and WBC is buffer based and makes use of the fact that in mammalian blood, genomic DNA (gDNA) is only contained in the white cells (leukocytes). The Red cells (erythrocytes) and platelets lack a nucleus and so neither contains gDNA. (bird erythrocytes are an exception) In general, the average number of white cells in 1 mL of human blood is about 7 million.
The first step in our protocol uses a hypotonic lysis buffer that preferentially lyses RBC. The WBC are pelleted and then the heme containing RBC supernatant is removed. A chaotrophic buffer is added to the WBC pellet and the nucleic acids are released. As mentioned above, this is a good point to freeze the sample for future RNA isolation at (-20 C or -80 C) for long term storage if the sample can’t be processed right away. Our ClearMag® Beads are then used to capture the nucleic acids without binding unwanted contaminants. Subsequent washes and elution generate ready-to-use DNA and RNA for most any downstream application.
Average RNA and DNA yields?
Not all blood samples are the same. They differ in the number of WBCs and this affects the quantity of nucleic acids isolated from each sample. WBC count can vary based on the health of the subject at the time of blood sampling. Nucleic acid yields vary by species as well.
High Molecular Weight nucleic acids
If you are looking for high molecular weight DNA from blood in a high throughput manner, this kit is the way to go! We’ve been able to isolate up to 100kb fragments of DNA so if you are looking for targets that are low in abundance or need longer chunks of DNA for sequencing purposes, look to this blood kit!
With all of that said and the vampire in R&D, hunger is looming and its best to get back to the bench where my stock of holy water and garlic bulbs are awaiting my arrival. Later pumpkin skaters!
- 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.
- DNA isolation by a rapid method from human blood samples: effects of MgCl2, EDTA, storage time, and temperature on DNA yield and quality. Lahiri DK1, Schnabel B. Biochem Genet. 1993 Aug;31(7-8):321-8.