DNA is stable, so it is not a big deal to store such samples. You can well store the frozen EDTA blood samples. However, I would prefer to store the isolated and purified DNA. You could store it at room temp or at 4°C, but it is adviseable to freeze them for longer storage times (weeks, months, years). There is no special treatment required. Isolation and purification are most simple using column-based kits (just for instance the QIAgen Blood kit; there are many other companies selling such kits, and to my knowledge they are all good enough).

If you are freezing the blood samples: aliqout them in 200µl before freezing.

Store in freezer -80oC to keep the blood in fresh condition.

DNA is stable, so it is not a big deal to store such samples. You can well store the frozen EDTA blood samples. However, I would prefer to store the isolated and purified DNA. You could store it at room temp or at 4°C, but it is adviseable to freeze them for longer storage times (weeks, months, years). There is no special treatment required. Isolation and purification are most simple using column-based kits (just for instance the QIAgen Blood kit; there are many other companies selling such kits, and to my knowledge they are all good enough).

If you are freezing the blood samples: aliqout them in 200µl before freezing.

Thanks Shiau, will it require any pre treatments prior the PCR analysis or any specific way of bringing the sample down to RT for the subsequent PCR analysis?

Thanks Jochen, Appreciate your advice. I'm just thinking of storing EDTA blood (whichever the method that gives the best results) and then use the whole blood for PCR w/o purifying DNA. It seems, frozen EDTA blood would do the job, whereas I am not very certain about the exact way of bringing the sample to RT (if required). I've heard thawing has resulted disruptions to DNA and couldn't find any well written article on that. So do you have any experiences with using frozen blood for PCR earlier?

If you are going to perform an RT then you are analyzing RNA, not DNA, right? My advice was for DNA. However, for RNA I would recommend to purify the RNA and strore it this way. The blood cells contain RNAses that will in the long run degrade the RNA in the whole-blood sample. The precence of EDTA and low temperatures slow this process but do not prevent it. Further, freezing and subsequent thawing will disrupt cells so that the RNAses get better access to the RNA. Not good.

I don't have a protocol using blood samples without purification directly in RT reactions. I see two problems: intracellular RNA might not be (well) accessible and RNAses will degrade your RNA. If these are no real problems, you just have to make sure to add enough Mg++ to activate the RT enzyme (Mg++ is captured by EDTA).

It is 10min work to purify the RNA... if a recovery loss of 10-20% are not a problem I don't see a reason against a purification.

I would agree with Jochen, if your downstream applications involve the RNA, then I would purify from fresh whole blood and then store the isolated total RNA at -80 rather than wait to isolate it later. For DNA, there is no problem simply storing whole blood at -80 for many years with little effect on DNA stability...I do this routinely and never have issues for PCR, Taqman, etc.

Good luck!

Thanks Barry & Jochen, I meant room temperature by RT, not Reverse Transcription. Sorry about that. I guess I will have no problems of using frozen EDTA blood for PCR. May be I was wrong with the idea that thawing might disrupt the DNA. I am sorry my mole bio knowledge is below average. Just wanted to know if there is a specific way (minimizing the DNA disruption, if any) of thawing the frozen EDTA blood and then subject to PCR?

So you want to put a drop of blood directy into the PCR tube?

I would not do it.

I see the biggest problems in

i) blood contains potent PCR inhibitors, so the PCR might not work well anyway

ii) the DNA is in the chromatin in the nuclei and might not be well accessible even after some minuted boiling (initial denaturation phase in PCR).

Purification solves both problems efficiently.

Degradation of DNA because of freezing/thawing is not a big problem, in particular not for PCR. You don't require intact Mb-long fragments to amplify sequences of some 100 or 1000 bp. If you go for amplification of Mb-sequences, well, that's quite another problem! (I don't know anybody doing this and I don't now if this would be possible at all; I guess one would need to add helicases and pocessivity factors and a highly processive Pol with proof-reading and and and...) I guess you wish to do a normal, conventional PCR to detect some sequence or gene, as a preparation for a sequencing or something like that. In this case, degradation or fragmentation of the DNA (to a usual degree; I am surely not talking about near-total degradation!) as probably caused by freezing/thawing or pipetting is not at all problematic. In contrast, it might be advantagous because it may resolve steric problems of very high-molecular DNA.

So you can store the blood or the purified DNA. But always use purified DNA for PCR. I would store the blood at -80°C, at least for long-terms. The pure DNA can be stored easily for several month at 4°C - in case the room in freezers is limited.

Oh i see, yes Jochen is correct, you must purify the DNA prior to any kind of PCR due to the inhibitors present in red blood cells. You will have no problems storing whole blood for long periods of time at -80 until you're ready to do PCR. You will have to isolate the genomic DNA prior to attempting PCR obviously. There will be very little effect of freeze/thaw on the DNA stability for your purposes.

Good luck!

You can use the Paxgene DNA tube to preserve your DNA and subsequently purify it in a single-tube protocol.

Hi,

For long term storage I suggest FTA cards or similar:

http://www.whatman.com/FTANucleicAcidCollectionStorageandPurification.aspx

Thank you Jochen & Barry. I much appreciate your suggestions and I will purify DNA prior PCR. There was a recent article which really encouraged me to go for whole blood PCR which had shown results as good as purified DNA. But as both of you've mentioned, I'd stick in to the purified DNA.

If you do not have the blood samples yet, then it gives you some time to get prepared for the DNA extraction. This I mention because DNA tend to degrade over time. So once you have have the samples, first you should extract the DNA which can then be stored for no matter how long.

And as you mentioned about blood PCR, it would need additional reagents to suppress the inhibitory substances in blood (this reagent is available as a special cocktail that suppresses these inhibitors). There are also blood DNA PCR kits. And these contain reagents to disrupt the WBC and the DNA remains in the total blood which can then be frozen or freshly used for PCR (it is this DNA to which the reagents get access for amplification). For blood DNA PCR you would need a modified polymerase too. So unless you have access to all these, blood PCR is not a good idea.

So its best to extract the DNA using a kit or the traditional phenol-chloroform protocol (the latter is tedious). and proceed with the normal PCR.

Try to separate the PBMC within 4hrs of collection and store the pellet in -80oC for future isolation of DNA/RNA.

As u wish to use the DNA for PCR (and RNA is of no use to u), here are my suggestions:

If u collect the blood in EDTA and store at 80C, after thawing, some of the RBCs lyse and this may not be recommended if u work with the parasites like malaria babesia, mycoplasma, anaplasma, hemoproteus etc.

If you are using the conventional methods like Phenol-CHCL3 to isolate the DNA or the Qiagen blood-DNA, u can directly go forward immediately after thawing. except that u might incur slight loss of dna due to the nucleases , there is no other widely used method. the FTA cards can also be used but the volume of blood that can be blotted on them is very low (200ul to the max).

If u r well funded, u can also go for the TRI reagent protocol , remove the supernatant(RNA) after the first spin following lysis and then work with the interphase DNA isolation. The best part of this is that u can lyse the cells in the reagent and then store the lysate in -80C (till upto 2 yrs but less is preferred) and then isolate RNA or DNA whever u require.

Final point: If u have no other option than freezing the blood, then go for it and then thaw them later (but isolate immediately after the freeze-thaw). Then lyse in the buffer (either of the kit or traditional TES) and proceed with isolation. whatever method u use, quantitate the DNA post isolation for purity and yield.

GoodLuck

Cheers