It’s Like Getting RNA From a Blood Sample
So you have some blood stored in the -20C or -80C and you want to isolate RNA from the samples. If you wanted DNA, you would have many products to choose from. But for RNA, your choices are more limited.
Obtaining RNA From Frozen Blood is Difficult
Why is that? The reason is that most RNA from blood kits start out with a red blood cell (RBC) lysis step using a hypotonic lysis buffer. The result is a nice white blood cell pellet (WBC) that is easy to extract RNA from. However, if the sample has been frozen, the thawing process is going to cause more cell damage. Cells will lyse from the shearing forces of ice crystals and from the shock to the cell membrane, and some of those WBCs will be lost.
The BiOstic Blood RNA Isolation Kit
One way to reduce this loss is to use the BiOstic Blood RNA Isolation Kit. This protects the cell pellet by using only one RBC lysis step to avoid extended time and handling of the already compromised cells.
But what if the blood was frozen and thawed twice? This sample will have too much cell damage to perform an RBC lysis and will not yield usable RNA. In addition to the shearing, RNases are active and working in dead cells. Between the degradation and the loss of WBCs, your yield will be minimal.
So How Do You Get RNA From Blood?
Starting with fresh blood gives the best result. Don’t freeze it. It is better to store the blood at 4C for short amount of time (~ 1hr) if you can’t get to it immediately.
But a better stopping point is to perform the RBC lysis and then lyse the WBC pellet in a guanidine containing lysis buffer that will protect the RNA from RNases. The homogenized cells in guanidine will be stable stored at -20C until you can start again.
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 fresh.
What are My Other Options?
If processing your blood immediately is not a possibility, for example, if the blood comes from another location, then you may want to try one of the blood stabilization reagents available for protecting RNA profiles during storage.
Listed below are three examples of products used for storing blood before RNA extraction:
1. PAXgene Blood RNA Kit (QIAGEN)
The first system ever launched for stabilizing RNA from blood, the Qiagen PAXgene Blood RNA Tube (now sold by BD, purchase through VWR) has some unique advantages.
First, you can collect up to 2.5 ml of blood and store it for up to 5 days at room temperature and then purify RNA from the entire volume on a single spin filter using a standard laboratory microcentrifuge.
The reagent comes already aliquotted in a blood tube so you can get stabilization immediately as the blood exits the patient. And because this system has been out a long time, there are many references and strong data validating its use.
The main disadvantage of this system is cost. The tubes are very expensive (~$10 each) and so is the PAXgene Blood RNA purification kit ($8.12 a prep) .
An alternative kit for processing PAXgene Blood RNA Tube collected samples exists. However, because of the difficulty in handling this sample, standard RNA prep kits and TRIzol will not work. It has to be a product specifically designed to handle a PAXgene collected sample.
2. Tempus Blood RNA Tube (ABI/ Life Technologies)
The Tempus Blood RNA Tube from Applied Biosystems (now Life Technologies) was released after the PAXgene system and attempts to one-up Qiagen with several changes.
The amount of blood collected is 3 ml, direct into a vacutainer tube and the sample can be processed immediately or stored 5 days at room temperature. This is in contrast to the Qiagen kit which requires a minimum 2 hour incubation at room temperature before extracting the RNA.
But the cost per tube is much lower than the Qiagen kit at only $5.16 per Tempus Tube. But the RNA kit is $15 per prep when you buy the larger 24 prep kit.
Plus, it appears a specialized piece of equipment, an ABI PRISM 6100 Nucleic Acid PrepStation, is required to run this prep. So cost seems to be a disadvantage of the Tempus system.
More worrisome is this comment prior to step one of the protocol: “If you are processing a large batch of samples, keep the samples on ice as much as possible. Otherwise, RNA yields may decrease significantly.” This statement does not equate with a product that is supposed to stabilize RNA at room temperature for 5 days.
3. RNALater for Blood (Ambion/Life Technologies)
The Ambion RiboPure- Blood Kit for isolating RNA from RNALater stabilized blood is a less expensive alternative to the first two reagents discussed.
RNALater is added directly to 0.3-0.5 ml of blood (1.3 ml of RNALater) and then the sample can be stored up to 3 days at room temperature.
For RNA purification, all of the cells are pelleted out of the RNALater and used for lysis. The main advantage of this system is the cost savings of the stabilization solution.
RNALater is less than $1/ ml. However, the kit is $9.20 per extraction. If you wanted to perform 4-5 preps to obtain the equivalent amount of RNA as the PAXgene system, then the cost would be prohibitive.
The other difference is that it requires two purification methods to isolate the RNA; a phenol extraction and then a silica spin filter. While the website says the protocol takes <30 minutes, the flow chart on the Ambion page (bottom right) says it takes 45 minutes plus 15 minutes for DNase treatment. Still, this might be the shortest protocol of the three and allows all of the processing to occur in a microcentrifuge.
As you can see from these options, getting RNA intact from blood is no small feat and companies have spent a lot of time and research into finding ways to preserve RNA from degradation during storage. Blood is rich in protein and inhibitors, all of which need to be removed in the process of extracting the larger volumes of sample needed to obtain enough yield for gene expression profiling experiments.
In my experience, the best results and least expensive methods are always using fresh blood processed immediately. But stabilization methods exist and depending on what volume of blood you want to start with and how much you want to spend per prep, you can select the right option for you.
Let us know if you have any experience with the methods described above and how they worked for you.
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You can use the GeneJet (from thermo) with the Tempus tubes. No specialized equipment. One cold initial microfuge spin. Then pretty much same as PAXgene.
Paxgene kits user here – we get satisfactory RNA yields and eventual microarray data from blood samples. Biggest problem, actually, is convincing phlebotomists NOT to freeze samples immediately so that the kit reagents have time to work at room temp. We end up thawing and letting them sit in the lab 4+ hours before use.
Thanks for putting this up, it’s a great recap of the challenges.