DE10219117C1 - Use of lithium dodecyl sulfate for stabilizing RNA in solution, particularly during purification of RNA from cell lysate - Google Patents

Abstract

Use of lithium dodecyl sulfate (I) for stabilizing RNA in solution. An Independent claim is also included for a method of stabilizing RNA in a (biological) sample, samples containing animal and/or human cells, and/or in blood by adding a stabilizing reagent that contains (I).

Description

The present invention relates to a ver drive to stabilize ribonucleic acids and Uses of stabilization buffers. such Procedures are used in the purification of Ribonuk linseed acids (RNA) needed.

Ribonucleic acids have a high level of information content and are more often the subject of molecular organic logical diagnostics. Unlike DNA, RNS is much more unstable and therefore more difficult to handle (Pasloske, 2001: Methods in Molecular Biology, vol 160, 105-111).

RNA degradation due to ubiquitous RNA before, during and after isolation of RNA a big problem because the integrity of the RNA Prerequisite for molecular biological diagnostics  is.

Studies have shown that RNA from human whole blood within a few hours of taking the blood is significantly degraded (McFaul et al., 2000, J. Lab. Clin Med., 135 (3), 263-269) what the possible capabilities for sample transport are very tight. The degradation of the RNA can be too nega tive results lead to the reduction by degradation transcript number in vitro with the transcript number not correlated in vivo.

Inhibiting RNA integrity is more endogenous RNase during cell lysis and avoid con contaminations with exogenous RNAsen during the RNS Insulation essential.

RNAsenes can be inhibited 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 both cause cell lysis as well as the inactivation of RNAsen.
• - Vanadyl ribonucleoside complexes (VRC): bind wide range of RNAsen, but inhibit but also other enzymes (see below)
• - complexing agents: e.g. B. Solids such as Ben tonite, macaloid, which bind the RNAsen and thereby separate from the RNAsen in solution NEN.
• - enzymes:
  Proteinase K digests cell proteins and is very active in the presence of Na dodecyl sulfate (SDS) at 65 ° C (1);
  RNAse inhibitor from human placenta, rnasin: they only protect the RNA from degradation under non-denaturing conditions (Murphy et al., 1995: BioTechniques, Vol. 18, No. 6, 1068-1073).

All of these reagents have strong limitations: ge ring spectrum of RNAsen activity, additional inhibition enzymes used for the subsequent detection of RNA are essential (VRC's inhibit RNA polymerases and in vitro transcription / translation (Pasloske, 2001: Methods in Molecular Biology, vol. 160, 105-111), Not applicable during cell lysis (RNAsin and other enzymatic RNAse inhibitors are just under active and suitable for non-denaturing conditions this does not signify inhibition during the first RNA Purification Steps).

The object of the present invention is a ver Improved stabilization process for ribonuclein specify acids.

This task is accomplished through the use of lithium Dodecyl sulfate according to claim 1 and the method solved according to claim 10. Advantageous further training the use according to the invention and the inventions The method according to the invention is in the respective pending claims.

The basis of the present invention is the cognition nis that lithium dodecyl sulfate stabilization caused by RNA in solution. It could be more surprising be shown to show the RNA by adding Lithium dodecyl sulfate for many hours to days is stabilized, although in the respective solution RNases  were present. This is particularly important Role when animal cells, for example, by Ly se or osmotic shock are unlocked and the RNS and RNAsen were present together in the solution gene.

Stabilization is particularly advantageous solution containing lithium dodecyl sulfate of 0.1% (w / v) to 5% (w / v).

It is particularly advantageous if the stabilization approx. 1% (w / v) lithium dodecyl sulfate contains.

Furthermore, the stabilizing effect is under supports if the salt lithium chloride (LiCl) of the Sta bilizing solution is added, advantageous Wei se with a concentration between 100 mmol / l and 2 mol / l.

A further support of the stabilizing effect is by adding dithiothreitol (DTT).

The following is an example of how to do this given an RNA stabilization according to the invention.

Fig. 1 shows the results of a measurement on freshly prepared RNA as well as after hour 3, 24 hours and 48 hours incubation in the OF INVENTION to the invention stabilizing solution. In each case, a total of 4 samples were examined which contained no tumor cells, 10 tumor cells, 100 tumor cells or 1000 tumor cells per ml blood sample.

For this purpose, a defined number of tumor cells (0/10/100/1000 cells) in 1 ml of blood each  healthy control person inoculated with the help of anti body-coupled magnetic particles of the not bin end blood components separated and in each 100 µl lysis buffer (50 mM Tris-HCl pH 7.5; 0.5% (Vol / Vol) Triton X100) added. The separated Cells are lysed on the particles.

After the immunomagnetic particles have been separated off, 100 μl of the RNA-stabilizing buffer are contained
150 mmol / l Tris-HCl pH 7.5
1.5 mol / l LiCl
20 mmol / l EDTA pH 8.0
2% (w / v) Li dodecyl sulfate
10 mmol / l dithiothreitol
added. This solution was incubated for various times (0, 3, 24 and 48 hours) at room temperature. This was followed by mRNA isolation using oligo (dT) ₂₅ coupled magnetic particles (Dynabeads from Dynal). The processing was carried out according to the protocol in the Dynal manual. The lysate was incubated with the magnetic particles (20 ul Oli go (dT) _25- Dynabeads per batch) for 10 minutes at room temperature while rotating on the overhead shaker. This is 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% (w / v) Li dodecyl sulfate
B:
10 mmol / l Tris-HCl pH 7.5
0.15 mol / l LiCl
1 mmol / l EDTA ph 8.0

After the first wash step with wash buffer B is  the reaction solution into a new reaction vessel leads. Then the one bound to the magnetic particles mRNA with 100 µl ice-cold 10 mmol / l Tris-HCl solution washed and re in 29.5 ul RNAse-free water suspended. After 5 minutes of incubation at 50 ° C and After shaking gently, a conventional one was used cDNA synthesis. A polymerase kit was then used ten reaction (PCR) with tumor marker-specific primers carried out. The amplificates were in the Agilent Bioa nalyzer 2100 separated and visualized. The Ver Reverse transcription and amplification steps They were made under the descriptions below conditions:

Reverse transcription

The cDNA synthesis took place at 37 ° C for 1 hour subsequent inactivation of the reverse transcript se for. 5 minutes at 93 ° C and cooling on ice. (Sensiscript ™ Reverse Transcriptase Kit; Qiagen, Hilden)

Table 1

Components of cDNA synthesis

amplification

The polymerase chain reaction (PCR) was carried out with tumor marker-specific primers performed.  

PCR primers used

CK20 sense T60: ATC TCC AAG GCC TGA ATA AGG TCT
CK20 antisense T61: CCT CAG TTC CTT TTA ATT CTT CAG T

Table 2

Components of the PCR reaction

Table 3

PCR conditions

1 µl of the respective PCR approach was in the Agilent Bi oanalyzer 2100 separated on a DNA chip (500) and electronically documented the separation result.

Fig. 1 now shows in lane 1 a ladder of markers for the molecular weight. Lanes 2-5, 6-9, 10-13, 14-17 correspond to the samples that were incubated 0, 3, 24, 48 hours before the isolation of the RNA in the stabilizing solution. In each of these groups of lanes, one lane shows the sample with 0, 10, 100 or 1000 tumor cells in the starting blood sample, as labeled above the figure.

It can be seen immediately that the arrow drew tumor markers clearly and in almost unchanged form intensity at 0, 3 and 24 hours, respectively bated samples in each of the samples, the tumor cells contained, can be recognized. Only for the 48 hours the incubated samples is for the sample with 10 Tu morcell in the initial blood sample no tumor marker Detect signal (band). Obviously, here there was little degradation of RNA. Still is in the blood samples with 100 or 1000 tumor cells Tumor markers can still be clearly demonstrated. So that is ge shows that the stabilizing solution causes over at least 1 day and with appropriate RNS Concentration 2 days the RNA in the stabilization solution is broken down or extremely slowly.

Claims (21)

1. Use of lithium dodecyl sulfate for stabilization RNAization in a solution. 2. Use according to the preceding claim Stabilization of RNA in a biological Sample. 3. Use according to the preceding claim Stabilization of RNA in a cell disruption. 4. Use according to the preceding claim Stabilization of RNA in a cell disruption animal or human cells. 5. Use according to one of the preceding claims to stabilize RNA in a blood sample be. 6. Use according to one of the preceding claims che in a concentration of 0.1% to 5% (w / v) Li dodecyl sulfate. 7. Use according to one of the preceding claims surface in a concentration of 2% (w / v) li Dodecyl sulfate. 8. Use according to claim 6, characterized 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, characterized 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. Procedure for stabilizing RNA in one Sample, in biological samples, containing samples animal and / or human cells and / or in blood samples, characterized in that ver the sample with a stabilizing solution is set, which contains lithium dodecyl sulfate. 11. The method according to the preceding claim, since characterized by that the sample with a Stabilizing solution is added, the 0.1% contains up to 5% (w / v) lithium dodecyl sulfate. 12. Procedure according to one of the two previous Claims, characterized in that the Pro be mixed with a stabilizing solution is, the 2% (w / v) lithium dodecyl sulfate contains.   13. The method according to any one of claims 10 to 12, characterized in that the sample is mixed with a stabilizing solution, the
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
Contains 1-50 mmol / l dithiothreitol. 14. The method according to claim 12, characterized in that the sample is mixed with a stabilizing solution
150 mmol / l Tris-HCl pH 7.5 and / or
1.5 mmol / l LiCl and / or
20 mmol / l EDTA and / or
Contains 10 mmol / l dithiothreitol. 15. The method according to any one of claims 10 to 14, characterized in that the RNS isolated from the sample, cleaned up or fixed is enriched. 16. The method according to any one of claims 10 to 15, characterized in that after adding the stabilizing solution free RNA bound to a substrate, this min washed once and then the RNA is detached from the substrate. 17. The method according to the preceding claim, since characterized in that as a substrate magneti cal particles are used. 18. The method according to any one of claims 16 or 17, characterized in that the RNA is detached from the magnetic particles by means of a 10 mmol / l Tris-HCl solution in H ₂ O, pH 7.5. 19. The method according to any one of claims 16 to 18, characterized in that the RNS after the Detachment from the substrate into an RNAse-free one Buffer is added. 20. The method according to any one of claims 10 to 19, characterized in that RNA-containing compo be removed from the sample before the stabilizing solution to the separated Components is added. 21. The method according to any one of claims 10 to 20, characterized in that cells from a Blood sample to be separated before the Stabili Solution added to the separated cells will give.