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(12) Patent Application Publication (10) Pub. No.: US 2015/0225712 A1 Ginther Et Al

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(12) Patent Application Publication (10) Pub. No.: US 2015/0225712 A1 Ginther Et Al US 20150225712A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2015/0225712 A1 Ginther et al. (43) Pub. Date: Aug. 13, 2015 (54) METHOD FOR ISOLATING NUCLEICACIDS Publication Classification FROMA FORMALDEHYDE RELEASER STABILIZED SAMPLE (51) Int. Cl. CI2N 5/10 (2006.01) (71) Applicant: QIAGEN GmbH, Hilden (DE) CI2O I/68 (2006.01) (72) Inventors: Kalle Ginther, Hilden (DE); Ralf (52) U.S. Cl. Wyrich, Hilden (DE); Uwe Oelmiller, CPC .......... CI2N 15/1003 (2013.01); C12O I/6806 Hilden (DE) (2013.01) (21) Appl. No.: 14/423,062 (57) ABSTRACT (22) PCT Filed: Aug. 21, 2013 (86). PCT No.: PCT/EP2013/067351 The present invention pertains to a method for isolation and S371 (c)(1), purification of nucleic acids from a stabilized sample or por (2) Date: Feb. 20, 2015 tion or fraction thereof, wherein the sample stabilization involved the use of at least one formaldehyde releaser and (30) Foreign Application Priority Data wherein the isolation of the nucleic acids from the stabilized sample or portion or fraction thereof involves the use of at Aug. 21, 2012 (EP) .................................. 12181137.6 least one cationic detergent during lysis. Patent Application Publication Aug. 13, 2015 Sheet 1 of 8 US 2015/0225.712 A1 Figure 1a ? ? FIÑA Figure 1b 3.2 3.0 2.8 2.6 2.4 2.2 2.0 1.8 1.6 1.4 1.2 1.0 0.8 0.6 2 r1 r EDTA STRECK tO Patent Application Publication Aug. 13, 2015 Sheet 2 of 8 US 2015/0225.712 A1 Figure 2 [7] ºlae E=r='), E==: Patent Application Publication Aug. 13, 2015 Sheet 3 of 8 US 2015/0225.712 A1 Figure 3a §§ § ? §§ §§§ § §§§ § §§ |ssssssssssss§ § § §. §§ §§ §§ § §. §§ §§ §§§§ § §§§§ § §§ B='<<<<< Figure 3b 2.8 tO t24 t72 tO t24. OAzol TDTMA EDTA Patent Application Publication Aug. 13, 2015 Sheet 4 of 8 US 2015/0225.712 A1 Figure 4a TIÑ$??????????????????? Figure 4b No. FIFIIODOTTFIFTIFT ?l?L?l????????????? 55, FIFIIODOTTFIFTIFT FIFTFIFTFIFTFIIFIDOD FIFIIODOTTFIFTIFT §§ 55, Patent Application Publication Aug. 13, 2015 Sheet 5 of 8 US 2015/0225.712 A1 Figure 4c INo.. ? §§§§§ I-IIae. $$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$ Patent Application Publication Aug. 13, 2015 Sheet 6 of 8 US 2015/0225.712 A1 Figure 5a E. tounsiencºl-o*c)ºpºfQiçNo. % EE 2 § § |- § ||- Figure 5b Ntounsico+oc)ºpºfinopNo. TDTMA TDTMA PAXP OAzo EDTA PAXT EDTAC Patent Application Publication Aug. 13, 2015 Sheet 7 of 8 US 2015/0225.712 A1 Figure 6 g s is: :- -) control 3 (-) control Calibration EDTA + a. dest) (PAXgene) control Patent Application Publication US 2015/0225.712 A1 Figure 7a Figure 7b 2.4 2.O 1.6 1.2 0.8 0.4 O.O US 2015/0225.712 A1 Aug. 13, 2015 METHOD FOR ISOLATING NUCLECACDS 0005 Similar stabilisation agents that achieve an immedi FROMA FORMALDEHYDE RELEASER ate lysis of the sample are sold by ABI/Life Technologies with STABILIZED SAMPLE the Tempus Blood RNA tube product. The tubes also com prise a stabilisation reagent and the sample, here blood, that is 0001. The work leading to this invention has received drawn into the tube and mixed with this stabilization reagent funding from the European Community’s Seventh Frame is immediately lysed and cellular RNases are inactivated and work Programme (FP7/2007-2013) under grant agreement nucleic acids are precipitated. The vast majority of proteins no. 222916. remains in solution. FIELD OF THE INVENTION 0006. The disadvantage of the respective methods is that the stabilisation results in the complete lysis of the cells, 0002 This invention pertains to the isolation of nucleic thereby destroying the cells morphology. However, not only acids from samples that were stabilised using a formaldehyde the quality and quantity of the isolated nucleic acids respec releaser. tively their expression profile is of analytical interest, but also the presence, absence or number of specific cells contained in BACKGROUND OF THE INVENTION the sample Such as for example a blood sample. The destruc 0003 Nucleic acids are important biomarkers in the diag tion of the cells is a great disadvantage because any cell nostic field. Profiles of transcripts of the genome (in particular sorting or cell enrichment respectively cell analysis becomes mRNA and miRNA) are widely used as biomarkers in impossible. This is a disadvantage with respect to the sensi molecular in vitro diagnostics and promised to provide inside tivity of the detection of for example, circulating tumor cells into normal biological and pathological processes with the in the given background of normal white blood cells. hope of predicting disease outcome and indicating individu 0007. Therefore, very often specific stabilisation reagents, alised courses of therapy. Therefore, profiling of nucleic respectively blood collection tubes are provided that are spe acids, in particular RNA, is becoming important in disease cifically intended for the stabilisation of cells. The respective diagnosis, prognosis and in clinical trials for biomarker dis products allow to investigate the cellular content of the covery. The ability to obtain quantitative information from sample after storage, for example to detect the presence of the transcriptional profile would thus be a powerful tool to tumor cells for example by fluorescence activated cell sorting explore basic biology, diagnose disease, facilitate drug devel (FACS) analysis or changes of the ratio of different white opment, tailor therapeutics to specific pathologies and blood cells to each other by flow cytometry (FC) or FACS genetic profiles and also to generate databases relevant to analysis. E.g. many workflows use standard EDTA blood biological or therapeutic processes and pathways. Significant collection tubes for flow cytometry or FACS analysis, improvements of downstream assays and data analyses (ana although blood cells show minor lysis over time of storage. A lytical process) have been made during the last years. How further product from Streck Inc. is a direct-draw vacuum ever, it was found that the preanalytical steps, such as sample blood collection tube for the preservation of whole blood stabilisation, in particular for new biomolecular targets, have samples for immunophenotyping by flow cytometry. It pre a severe impact on the expression profile and may compro serves white blood cell antigens allowing Subsets of leuco mise the Subsequent analysis (see for example Hartel et al. cytes to be distinguished by flow cytometry analysis. The 2001, Pahl and Brune, 2002). Without precaution in the sta technology to maintain the integrity of the white blood cell bilisation of the sample to be analysed, the sample will cluster of differentiation (CD) markers is e.g. covered by U.S. undergo changes during transport and storage that may alter Pat. No. 5,460,797 and U.S. Pat. No. 5,459,073. the expression profile of the targeted molecules (see for 0008. However, using different stabilisation reagents and example Rainen et al., 2002; Baechler et al., 2004). If the accordingly stabilisation tubes for collecting the sample for expression profile is altered due to the handling of the sample, nucleic acid analysis and cell analysis is tedious. There is a the Subsequent analysis does not reflect the original situation need to reduce the number of different sample collection of the sample and hence of the patient but rather measure an tubes, for example blood collection tubes, per draw at the artificial profile generated during sample handling, transport patients’ site that are dedicated to different downstream and storage. Therefore, optimized stabilisation processes are assays (e.g. detection of cells and analysis of RNA). needed which stabilise the expression profile. 0009. Therefore, sample collection and stabilisation sys 0004 Stabilisation of samples such as in particular blood tems are needed, which keep the cell's morphology intact samples of a longer period was formally performed with the while at the same time stabilising the nucleic acids. Respec addition of organic solvents such as phenol and/or chloroform tive stabilisation systems are advantageous for example in the or by direct freezing in liquid nitrogen or using dry ice. These molecular diagnostic of cancer, because they would make an methods are not at all practicable techniques for hospitals, enrichment of cells prior to the extraction of the nucleic acids doctor Surgeries or diagnostic routine laboratories. To over from the cells possible and would thereby increase the chance come these problems, PreAnalytiX developed the first to detect rare events of circulating tumor cells in the samples, research product for the collection of human blood with an for example a blood sample. This would increase the chance evacuated blood collection tube that contains reagents for an that a specific biomarker is identified in the sample. immediate stabilisation of the RNA gene expression profile at 0010. To address the need of simultaneous cell stabilisa the point of sample collection. The respective stabilisation tion and nucleic acid stabilisation, stabilisation systems were composition allows the transport and storage at room tem developed that are based on the use of formaldehyde releas perature without the risk of changes in the RNA profile by ers. Respective stabilisation agents are commercially avail gene induction and transcript degradation (see for example able from Streck Inc. under the name of cell-free RNABCT U.S. Pat. No. 6,617,170, U.S. Pat. No. 7,270,953, Kruhoffer (blood collection tube). The 10 ml blood collection tube is et al., 2007). The respective compositions are sold under the intended for the preservation and stabilisation of cell-free name of PAXgene Blood RNA Tubes. RNA in plasma for up to 3 days at room temperature. The US 2015/0225.712 A1 Aug. 13, 2015 preservative stabilizes cell-free RNA in plasma and prevents DETAILED DESCRIPTION OF THIS INVENTION the release of non-target background RNA from blood cells 0018. The present invention pertains to the use of at least during sample processing and storage.
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