Container for nucleic acid analysis
A nucleic acid analysis and container technology, applied in the field of blood samples, which can solve the problems that nucleic acid cannot be directly separated by solid phase, cannot be guaranteed, and nucleic acid degradation cannot be controlled.
- Summary
- Abstract
- Description
- Claims
- Application Information
AI Technical Summary
Problems solved by technology
Method used
Image
Examples
example 1
[0061] blood collection system
[0062] To have the above advantages, the blood collection system should consist of the following: a small test tube filled with a fixed-volume nucleic acid stabilization solution, in which there are also nucleic acid-connected solid-phase substances and a certain volume of vacuum, and sealed with a septum. Constructing this membrane should be compatible with current sample collection kits (cannula, etc.). Using the existing example: prepare 2.2ml of reagents in advance, adjust the vacuum volume so that 2.2ml of blood flows in during the sample collection process, and the nucleic acid in the blood is immediately converted into a stable form after the blood flows into the test tube.
[0063] Evaluate the following examples:
[0064] If not specifically mentioned, in all examples the nucleic acid stabilizing substance (N-sS) consisted of: 45 mM tris, 5 M guanidine dithiocyanate, 0.8% (w / v) dithriothreitol, 18% (w / v) Triton -X-100, pH 6.0.
[00...
example 2
[0068] Stability of nucleic acids after mixing samples with N-sS
[0069] Isolation of DNA and RNA from sample lysates using silica-derived particles.
[0070] Materials and methods
[0071] After the sample material was collected, it was stored at 4°C for 6 days and at -20°C for 1 month, and then the DNA and RNA were separated. RNA isolation can be performed using a high-purity RNA isolation kit (Boehringer Mannheim, Cat-No 1808665). The steps described in the instructions were modified as follows: add 2.4ml of sample lysate to 4 equal volumes of 600ul on the column, so that 2.4ml of sample lysate was used, and RNA was finally eluted with 100ul of elution buffer.
[0072] Use the QiaAmp Blood kit (Qiagen-Cat-No 29104) to separate DNA (Figure 3), and modify the standard steps described in the instructions at different points: 400ul samples are directly loaded on the column, and the binding agent in the kit is not used , add 25ul proteinase K digestion solution, incubate at ...
example 3
[0074] Importance of reducing agents such as DTT in stabilizers for long-term stability of RNA
[0075] Materials and methods
[0076] Available stabilizing solutions:
[0077] 4.0MGTC; 13.5% Triton-X-100, 45mMtris / HCl, 120mMDTT (optional), 700ul of serum and 700ul of stable solution were mixed, incubated for 2 minutes and then added with MS2-RNA (0.8ug / ul, purchased from RocheDiagnostics), Incubate at 40°C for 180 minutes, then divide it into 400ul aliquots and react with the high-purity RNA kit (Roche) according to experiment 1, elute the sample with 50ul eluent, freeze at -20°C, and pass through agarose gel Analysis proved effective.
[0078] Conclusion: Long-term stability of RNA cannot be obtained without adding reducing agent in the stabilizer.
PUM
Login to View More Abstract
Description
Claims
Application Information
Login to View More