Stabilization and isolation of extracellular nucleic acids

Abstract

The present invention provides methods, compositions and devices for stabilizing the extracellular nucleic acid population in a cell-containing biological sample using butanamide.

Description

[0001]The work leading to this invention has received funding from the European Community's Seventh Framework Programme (FP7 / 2007-2013) under grant agreement n° 222916.FIELD OF THE INVENTION[0002]The technology disclosed herein relates to methods and compositions suitable for stabilizing the extracellular nucleic acid population in a cell-containing sample, in particular a blood sample, and to a method for isolating extracellular nucleic acids from respectively stabilized biological samples.BACKGROUND[0003]Extracellular nucleic acids have been identified in blood, plasma, serum and other body fluids. Extracellular nucleic acids that are found in respective samples are to a certain extent degradation resistant due to the fact that they are protected from nucleases (e.g. because they are secreted in form of a proteolipid complex, are associated with proteins or are contained in vesicles). The presence of elevated levels of extracellular nucleic acids such as DNA and / or RNA in many med...

AI Technical Summary

Benefits of technology

The patent describes methods and compositions for stabilizing biological samples, particularly cell-containing samples, to prevent contamination and degradation of extracellular nucleic acids. The methods involve contacting the sample with stabilizing agents that reduce cell lysis and release of genomic DNA from cells. The stabilized sample can then be stored and transported for further analysis without risking contamination. The stabilization technology does not require the use of cross-linking reagents, which can lead to reduced recovery of nucleic acids. The patent also describes the use of certain stabilizing agents that are effective in stabilizing the transcriptome of contained cells. The invention has advantages over existing stabilization methods that use cross-linking reagents. The patent also mentions the use of solutions and buffers containing stabilizing agents.

Problems solved by technology

However, obtaining an essentially cell-free fraction of a sample can be problematic and the separation is frequently a tedious and time consuming multi-step process as it is important to use carefully controlled conditions to prevent cell breakage during centrifugation which could contaminate the extracellular nucleic acids with cellular nucleic acids released during breakage.

Furthermore, it is often difficult to remove all cells.

Once cell lysis begins, the lysed cells release large amounts of additional nucleic acids which become mixed with the extracellular nucleic acids and it becomes increasingly difficult to recover the extracellular nucleic acids for testing.

Further, the amount and recoverability of available extracellular nucleic acids can decrease substantially over a period of time due to degradation.

However, as discussed above, a major problem regarding the analysis of circulating, cell-free nucleic acids (cfNA) from tumors or of foetal origin is—besides the degradation that occurs in serum and probably also plasma—the possible dilution of extracellular DNA (and RNA) by genetic material from damaged or decaying blood cells after blood collection.

In particular the lysis of white blood cells is a problem as they release large amounts of genomic DNA in addition to RNA.

The above discussed problems particularly are an issue, if the sample comprises a high amount of cells as is the case e.g. with whole blood samples.

However, the need to directly separate e.g. the plasma from the blood is a major disadvantage because many facilities wherein the blood is drawn (e.g. a doctor's practice) do not have a centrifuge that would enable the efficient separation of blood plasma.

Furthermore, plasma that is obtained under regular conditions often comprises residual amounts of cells which accordingly, may also become damaged or may die during handling of the sample, thereby releasing intracellular nucleic acids, in particular genomic DNA, as is described above.

These remaining cells also pose a risk that they become damaged during the handling so that their nucleic acid content, particularly genomic (nuclear) DNA and cytoplasmic RNA, would merge with and thereby contaminate respectively dilute the extracellular, circulating nucleic acid fraction.

However, again, such powerful centrifuges are often not available at the facilities wherein the blood is obtained.

This too imposes practical constraints upon the processing of the samples as e.g. the plasma samples must be shipped frozen.

This increases the costs and furthermore, poses a risk that the sample gets compromised in case the cold chain is interrupted.

However, even though EDTA is an efficient anticoagulant, EDTA does not efficiently prevent the dilution respectively contamination of the extracellular nucleic acid population by released intracellular nucleic acids during storage.

Thus, the extracellular nucleic acid population that is found in the cell-free portion of EDTA stabilised samples changes during the storage and becomes contaminated with large amounts of intracellular nucleic acids, in particular genomic DNA.

Accordingly, EDTA is not capable of sufficiently stabilizing the extracellular nucleic acid population in particular because it can not avoid the contamination of the extracellular nucleic acid population with e.g. genomic DNA fragments which are generated after blood draw by cell degradation and cell instability during sample transportation and storage.

However, these methods are based on the immediate lysis of the cells contained in the sample.

Therefore, these methods and other methods that induce cell lysis are unsuitable for stabilizing the extracellular nucleic acid population in a cell-containing sample, because they induce the release of intracellular nucleic acids which become thereby mixed with the extracellular nucleic acid population.

However, N,N-dimetyhlacetamide is a toxic agent.

However, the use of formaldehyde or formaldehyde-releasing substances has drawbacks, as they compromise the efficacy of extracellular nucleic acid isolation by induction of crosslinks between nucleic acid molecules or between proteins and nucleic acids.

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