DE 102 19 117 C 1
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`(12) Patent Specification (51) Int. Cl.7:
`(19) FEDERAL REPUBLIC OF
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` GERMANY (10) DE 102 19 117 C 1 C 12 N 15/10
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`(21) Reference:
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`102 19 117.4-41
`(22) Application date:
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`29/04/2002
`(43) Publication date:
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`(45) Publication date of patent grant: 30/10/2003
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` GERMAN
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` PATENT AND
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` TRADEMARK OFFICE
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`Objections can be made within 3 months of the publication of the grant
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` (72) Inventors:
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`(73) Patent proprietor:
` AdnaGen AG, 30853 Langenhagen, DE
` Stefan, Monica, Dr., 30173 Hanover, DE; Krehan,
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` Alf-Andreas, Dr., 30853 Langenhagen, DE; Böcher,
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` Oliver, Dr., 30853 Langenhagen, DE; Waschütze,
`(74) Agent:
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` Stefanie, Dr., 30169 Hanover, DE
` PFENNING MEINIG & PARTNER GbR, 80336
`(56) Publications considered for the assessment of
` Munich
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` patentability:
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` BIOSIS:AN:1987:48740, in Biochem. Biophys.
` Acta, 1986, 851 (3), pp. 395-406;
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`(54) Method for stabilising RNA and uses of stabilisation buffers
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`(57) The present invention relates to a method for stabilising ribonucleic acids and uses of
`stabilisation buffers. Such methods are required for the purification of ribonucleic acids.
`According to the present invention, a stabilising solution containing lithium dodecyl sulfate is added
`to a sample containing ribonucleic acid. As a result, the ribonucleic acid is stabilised for many hours
`to days, although ribonucleic acid-cleaving enzymes may also be present in the respective sample.
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`FEDERAL PRINTING OFFICE 09.03 203 440/210/7A 1
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`DE 102 19 117 C 1
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`Page 1
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`Spectrum Ex. 1005
`IPR Petition - USP 10,000,795
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`

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`DE 102 10 117 C 1
`Description
`
`[0001] The present invention relates to a method for stabilising ribonucleic acids and uses of
`stabilisation buffers. Such methods are required for the purification of ribonucleic acids (RNA).
`
`[0002] Ribonucleic acids have a high level of information content and are more often the subject of
`molecular biological diagnostics. Unlike DNA, RNA is much more unstable and therefore more
`difficult to handle (Pasloske, 2001: Methods in Molecular Biology, vol. 160, 105-111).
`
`[0003] The RNA degradation by ubiquitous RNases before, during and after the isolation of RNA is a
`major problem, since the integrity of the RNA is a prerequisite for molecular biological diagnostics.
`
`[0004] Studies have shown that the RNA from human whole blood is significantly degraded within a
`few hours after blood collection (McFaul et al., 2000, J. Lab. Clin. Med., 135 (3), 263-269), which
`greatly restricts the possibilities for sample transport. The degradation of the RNA can lead to false
`negative results because the reduced number of transcripts in vitro due to degradation does not
`correlate with the number of transcripts in vivo.
`
`[0005] For the integrity of the RNA, the inhibition of endogenous RNases during the cell lysis and the
`avoidance of contamination with exogenous RNases during the RNA isolation are essential.
`
`[0006] The inhibition of RNases can be achieved by adding chemical agents or some enzymes:
`- Strongly denaturing agents such as 8 M urea, 4 mol/l guanidinium hydrochloride, 4 mol/l
`guanidinium isothiocyanate cause both the lysis of the cell and the inactivation of RNases.
`- Vanadyl ribonucleoside complexes (VRC): bind a wide range of RNases, but also inhibit
`other enzymes (see below)
`- Complexing agents: e.g. solids such as bentonite, macaloid, which bind he RNases and
`thereby separate them from the RNases in solutions
`- Enzymes:
`Proteinase K digests cell proteins and is very active in the presence of sodium dodecyl sulfate
`(SDS) at 65°C (1); RNase inhibitor from human placenta, Rnasin: they protect the RNA from
`degradation only under non-denaturing conditions (Murphy et al., 1995: BioTechniques, Vol.
`18, No. 6, 1068-1073).
`
`
`[0007] All of these reagents have severe limitations: low RNases activity spectrum, inhibition of
`additional enzymes that are essential for the subsequent detection of RNA (VRC’s inhibit RNA
`polymerases and in vitro transcription/translation (Pasloske, 2001: Methods in Molecular Biology,
`vol. 160, 105-111), inapplicable speed during cell lysis (RNAsin and other enzymatic RNase inhibitors
`are only active under non-denaturing conditions and are therefore not suitable for inhibition during
`the first steps in RNA purification).
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`[0008] The object of the present invention is to provide an improved stabilisation method for
`ribonucleic acids.
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`DE 102 10 117 C 1
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`[0009] This object is achieved by the use of lithium dodecyl sulfate according to claim 1 and the
`method according to claim 10. Advantageous further developments of the use according to the
`invention and of the method according to the invention are given in the respective dependent
`claims.
`
`[0010] The present invention is based on the finding that lithium dodecyl sulfate stabilises RNA in
`solution. It could surprisingly be shown that the RNA is stabilised by adding lithium dodecyl sulfate
`for many hours to days, although RNases were present in the respective solution. This plays a role in
`particular when animal cells are opened up, for example by lysis or osmotic shock, and the RNA and
`RNases are present together in the solution.
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`[0011] A stabilising solution with a lithium dodecyl sulfate content of 0.1% (weight/volume) to 5%
`(weight/volume) is particularly advantageous.
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`[0012] It is particularly advantageous if the stabilising solution contains approx. 1% (weight/volume)
`lithium dodecyl sulfate.
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`[0013] Furthermore, the stabilising effect is supported if lithium chloride (LiCL) is added to the
`stabilising solution as a salt, advantageously at a concentration between 100 mmol/l and 2 mol/l.
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`[0014] The stabilising effect is further supported by adding dithiothreitol (DTT).
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`[0015] The following is an example of carrying out an RNA stabilisation according to the invention.
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`[0016] Fig. 1 shows the results of a measurement on freshly prepared RNA and after a 3-hour, 24-
`hour or 48-hour incubation in the stabilising solution according to the invention. In each case, a total
`of 4 samples were examined which contained no tumour cells, 10 tumour cells, 100 tumour cells or
`1000 tumour cells per ml of blood sample.
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`[0017] For this purpose, a defined number of tumour cells (0/10/100/1000 cells) were inoculated
`into 1 ml of blood from a healthy control person in each case, separated from the non-binding blood
`components using antibody-coupled magnetic particles and were taken up in 100 μl lysis buffer (50
`mM Tris-HCl pH 7.5; 0.5% (vol/vol) Triton X100). The separated cells are lysed on the particles.
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`[0018] After separating the immunomagnetic particles, 100 μl of the RNA-stabilising buffer
`containing
`150 mmol/l Tris-HCl pH 7.5
`1.5 mol/l LiCl
`20 mmol/l EDTA pH 8.0
`2% (weight/volume) lithium dodecyl sulfate
`10 mmol/l dithiothreitol
`are added. This solution was incubated for various times (0, 3, 24 and 48 hours) at room
`temperature. The mRNA was then isolated using oligo(dT)25-coupled magnetic particles (Dynabeads
`from Dynal). The processing was carried out in accordance with the protocol in the Dynal company
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`DE 102 10 117 C 1
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`manual. The lysate was incubated with the magnetic particles (20 μl oligo(dT)25 Dynabeads per
`batch) for 10 minutes at room temperature while rotating on the overhead shaker. This was then
`followed by 2 washing steps each with 100 μl washing buffer A or washing buffer B.
`A:
`10 mmol/l Tris-HCl pH 7.5
`0.15 mol/l LiCl
`1 mmol/l EDTA pH 8.0
`0.1% (weight/volume) lithium dodecyl sulfate
`B:
`10 mmol/l Tris-HCl pH 7.5
`0.15 mol/l LiCl
`1 mmol/l EDTA pH 8.0
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`[0019] After the first washing step with washing buffer B, the reaction solution is transferred to a
`new reaction vessel. The mRNA bound to the magnetic particles is then washed with 100 μl of ice-
`cold 10 mmol/l Tris-HCl solution and resuspended in 29.5 μl of RNAs-free water. After incubation for
`5 minutes at 50°C, with gentle shaking, conventional cDNA synthesis took place. A polymerase chain
`reaction (PCR) was then carried out with tumour marker-specific primers. The amplificates were
`separated and visualised in the Agilent Bioanalyzer 2100. The method steps of reverse transcription
`and amplification took place under the conditions described below:
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`Reverse transcription
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`[0020] The cDNA synthesis took place at 37°C for 1 hour with subsequent inactivation of the reverse
`transcriptase for 5 minutes at 93°C and cooling on ice. (SensiscriptTM Reverse Transcriptase Kit;
`Qiagen, Hilden)
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`Table 1
`Components of the cDNA synthesis
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`The cDNA synthesis took place in a 40 μl reaction batch
`Volume
`Final concentration
`29.5 μl
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`4 μl
`4 μl
`0.5 μl
`2 μl
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`1 x
`0.5 mmol/l in each case
`0.5 U
`4 U
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`Components
`RNA in water
`10 x RT buffer
`DNTP mix (every 5 mmol/l)
`RNase inhibitor
`Reverse transcriptase
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`Amplification
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`[0021] The polymerase chain reaction (PCR) was carried out with tumour marker-specific primers.
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`Used PCR primers
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`CK20 sense T60: ATC TCC AAG GCC TGA ATA AGG TCT
`CK20 antisense T61: CCT CAG TTC CTT TTA ATT CTT CAG T
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`DE 102 10 117 C 1
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`Table 2
`Components of the PCR reaction
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`Volume
`Components
`8 μl
`CDNA
`5 μl
`10 x PCR buffer*
`1 μl
`DNTP mix
`50 μmol in each case
`Primer
`0.5 μl
`Taq-DNA polymerase**
`ad 50 μl
`H2O
`(*contains 15 mmol/l MgCl2; **HotStarTaqTM DNA Polymerase; Qiagen, Hilden)
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`Final concentration
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`1 x
`200 μmol in each case
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`2.5 U
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`Table 3
`PCR conditions
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`95°C 15 min
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`94°C 1 min
`58°C 1 min
`72°C 1 min
`72°C 10 min
`4°C Pause
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`Pre-denaturation
`Cycle (35 cycles)
`1. Denaturation
`2. Annealing
`3. Extension
`Final extension
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`[0022] 1 μl of the respective PCR mixture was separated on a DNA chip (500) in the Agilent
`Bioanalyzer 2100 and the separation result was electronically documented.
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`[0023] Fig. 1 now shows in lane 1 a ladder made up of markers for the molecular weight. Lanes 2-5,
`6-9, 10-13 and 14-17 correspond to the samples that were incubated 0, 3, 24 and 48 hours before
`isolating the RNA in the stabilising solution. In each of these groups of lanes, one lane shows the
`sample with 0, 10, 100 or 1000 tumour cells in the starting blood sample, as is labelled above the
`figure.
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`[0024] It can be seen immediately that the tumour marker marked with an arrow can be seen clearly
`and with almost unchanged intensity in the samples incubated for 0, 3 and 24 hours in each of the
`samples which contained tumour cells. Only in the samples incubated for 48 hours is no tumour
`marker signal (band) to be seen for the sample with 10 tumour cells in the starting blood sample.
`Evidently, a slight breakdown of RNA has taken place here. Nevertheless, the tumour marker can still
`be clearly demonstrated in the blood samples with 100 or 1000 tumour cells. This shows that the
`stabilising solution has the effect that the RNA in the stabilising solution is not degraded or is
`extremely slowly degraded over at least 1 day and, if the RNA concentration is appropriate, for 2
`days.
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`Claims
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`1. Use of lithium dodecyl sulfate for stabilisation of RNA in a solution.
`2. Use according to the preceding claim for stabilisation of RNA in a biological sample.
`3. Use according to the preceding claim for stabilisation of RNA in a cell disruption.
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`DE 102 10 117 C 1
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`4. Use according to the preceding claim for stabilisation of RNA in a cell disruption of animal or
`human cells.
`5. Use according to any one of the preceding claims for stabilisation of RNA in a blood sample.
`6. Use according to any one of the preceding claims in a concentration of 0.1% to 5%
`(weight/volume) of lithium dodecyl sulfate.
`7. Use according to any one of the preceding claims in a concentration of 2% (weight/volume) of
`lithium dodecyl sulfate.
`8. Use according to claim 6, characterised in that the solution further contains the following
`components:
`10-500 mmol/l Tris-HCl pH 6.5-8.5 and/or
`100-3500 mmol/l LiCl and/or
`1-100 mmol/l EDTA and/or
`1-50 mmol/l dithiothreitol.
`9. Use according to claim 7, characterised in that the solution further contains the following
`components:
`150 mmol/l Tris-HCl pH 7.5 and/or
`1.5 mmol/l LiCl and/or
`20 mmol/l EDTA and/or
`10 mmol/l dithiothreitol.
`10. A method for stabilisation of RNA in a sample, in biological samples, samples containing animal
`and/or human cells and/or in blood samples, characterised in that the sample is mixed with a
`stabilising solution which contains lithium dodecyl sulfate.
`11. The method according to the preceding claim, characterised in that the sample is mixed with a
`stabilising solution which contains 0.1% to 5% (weight/volume) of lithium dodecyl sulfate.
`12. The method according to any one of the two preceding claims, characterised in that the sample
`is mixed with a stabilising solution which contains 2% (weight/volume) of lithium dodecyl sulfate.
`13. The method according to any one of claims 10 to 12, characterised in that the sample is mixed
`with a stabilising solution which contains
`10-500 mmol/l Tris-HCl pH 6.5-8.5 and/or
`100-3500 mmol/l LiCl and/or
`1-100 mmol/l EDTA and/or
`1-50 mmol/l dithiothreitol.
`14. The method according to claim 12, characterised in that the sample is mixed with a stabilising
`solution which contains
`150 mmol/l Tris-HCl pH 7.5 and/or
`1.5 mmol/l LiCl and/or
`20 mmol/l EDTA and/or
`10 mmol/l dithiothreitol.
`15. The method according to any one of claims 10 to 14, characterised in that the RNA is then
`isolated from the sample, purified or enriched.
`16. The method according to any one of claims 10 to 15, characterised in that, after the stabilising
`solution has been added, the free RNA is bound to a substrate, the latter is washed at least once,
`and then the RNA is detached from the substrate.
`17. The method according to the preceding claim, characterised in that magnetic particles are used
`as the substrate.
`18. The method according to any one of claims 16 or 17, characterised in that the RNA is detached
`from the particles using a 10 mmol/l Tris-HCl solution in H2O, pH 7.5.
`19. The method according to any one of claims 16 to 18, characterised in that the RNA is taken up in
`a RNase-free buffer after it has been detached from the substrate.
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`20. The method according to any one of claims 10 to 19, characterised in that components
`containing RNA are separated from the sample before the stabilising solution is added to the
`separated components.
`21. The method according to any one of claims 10 to 20, characterised in that cells are separated
`from a blood sample before the stabilising solution is added to the separated cells.
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`DRAWINGS PAGE 1
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`Number:
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`Int. Cl.7:
`Publication date:
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`DE 102 19 117 C1
`C 12 N 15/10
`30 October 2003
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`Tumour marker
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`203 440/210
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` 0 hours; 3hours; 24hours; 48hours;
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`UNITED STATES PATENT AND TRADEMARK OFFICE
`VERIFICATION OF A TRANSLATION
`
`I, the below named translator, hereby declare that:
`
`My nameandpost office address are as stated below:
`That I am knowledgeable in the English language and in the German language, andthat ]
`believe the English translation ofthe attached German document
`DE10219117C1(Stefan)is true and complete.
`I hereby declare that all statements made herein ofmy own knowledgeare true and that
`all statements made on information and beliefare believed to be true; and further that
`these statements were made with the knowledgethatwillful false statements and thelike
`so made are punishable byfine or imprisonment, or both, under Section 1001 of Title 18
`ofthe United States Code and, that such willful false statements mayjeopardize the
`validity ofthe application or any patent issued thereon.
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`Date: 26 May, 2021
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`Full nameofthe translator: Ian Nicholas Harper CLARK
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`Signatureofthe translator: Pan)
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`Post Office Address: Clayallee 333, 14169 Berlin, GERMANY
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