Simultaneous flow analysis of DNA and RNA
Simultaneous flow analysis of DNA and RNA
Simultaneous DNA and RNA analysis by flow cytometry provides information on the DNA content of the cell and the transcriptional status of the cell. This assay detects DNA and RNA by utilizing heterodimeric fluorescent substances that bind to DNA as well as heterodimeric fluorescent substances that electrostatically bind to single-stranded RNA.The primary requirement for this analysis is the activity of the cells, so freshly processed cells should be used. The simultaneous analysis of DNA/RNA by flow cytometry was originally used to classify and biologically characterize hematologic malignancies, including multiple myeloma.
Principle
The basic principle of simultaneous DNA and RNA analysis by flow cytometry is the detection of DNA and RNA through the use of heterodimeric fluorescent substances that bind to DNA and, by electrostatic interaction, to single-stranded RNA. the primary requirement for analysis is cellular activity, so freshly processed cells should be used.
Appliance
Simultaneous analysis of DNA/RNA using flow cytometry is commonly used to classify and biologically characterize hematologic malignancies, including multiple myeloma.
Operation method
Simultaneous flow analysis of DNA and RNA
Principle
The basic principle of simultaneous DNA and RNA analysis by flow cytometry is the detection of DNA and RNA through the use of heterodimeric fluorescent substances that bind to DNA and, by electrostatic interaction, to single-stranded RNA. the primary requirement for analysis is cellular activity, so freshly processed cells should be used.
Materials and Instruments
Equipment: Move The basic process of simultaneous flow analysis of DNA and RNA can be divided into the following steps: The following procedure should be carried out in disposable glass tubes at 0 ℃ to 4 ℃. 1.1 Adjust the concentration of the cell suspension to 1.0 × 106 cells/ml with PBS at a concentration of 2 mM MgCl2. Cells are fixed in ethanol or permeabilized with Triton X-100. 1.2 Place Solution A and AO working solution on ice. 1.3 Dispense 0.2 ml of freshly processed cell suspension into 12 mm × 75 mm one-time glass tubes. 1.4 Add 0.4 ml of Solution A and incubate for 45 s at 0 °C to 4 °C. 1.5 Add 1.2 ml of Solution A and AO Working Solution and place on ice. 1.5 Add 1.2 ml of AO working solution and analyze immediately. 1.6 Repeat steps 3-5 for each sample. 1.7 Rinse the flow cytometer with bleach and distilled water.
① flow cytometer
② Centrifuge
Reagents:
① 1 mg/ml AO: (Note: AO is a mutagen. (Note: AO is a mutagen. Its physicochemical and toxicological properties are not fully understood. (Note: AO is a mutagen and its physicochemical and toxicological properties are not well understood. Therefore, care should be taken when using it.) Weigh 50 mg of AO powder (Polysciences, Warrington, Pennsylvania; see Note 4.1) into a clean glass, add 50 ml of distilled water, seal with tinfoil, and stir until it dissolves (protect from light). Filter through No. 1 Waterman filter paper, wrap in tinfoil and store at 4 °C under low light, stable for 1 year (see Note 4.2).
② 0.2M citric acid: add 19.21 g of citric acid (Sigma, St. Louis, MO) to 500 ml of distilled water, stir until dissolved, store at 4 ℃, stable for 2 months.
③ 10 mM ethylenediaminetetraacetic acid (EDTA): add 2.17 g EDTA (GibcoTM Invitrogen, Carlsbad, CA) to 500 ml of distilled water, stir to dissolve, and store at 4 ℃ for 2 months.
④ The sample was prepared with 2 mM MgCl
2
PBS containing 2 mM MgCl2: Dilute 10 × calcium- and magnesium-free PBS (Irvine Scientific, Santa Ana, CA) to 1 ×. Then 0.408 ml per liter of 4.9 M MgCl
2
per liter. Adjust the pH to 7.2~7.4. 10 × stock solution can be stored for up to 3 months at room temperature. 1 × is stored at 4°C.
⑤ 1M sodium chloride (NaCl): add 29.0 g NaCl (Fisher, Houston, Houston) to 500 ml distilled water, shake to dissolve, store at 4 ℃ for 3 months.
0.4 M disodium hydrogen phosphate (Na
2
HPO
4
): add 28.39 g Na
2
HPO
4
(Fisher) to 500 ml of distilled water, shake to dissolve, and store at room temperature, 22 ℃, stable for 2 months.
⑦ 10X Triton: add 10 ml Triton X-100 (CMS, Houston, Houston) to 90 ml distilled water, dissolve by shaking, store at 4 ℃, stable for 3 months.
⑧ solution A: add 1 ml Triton X-100, 8 ml N HCL, 15 ml 1M NaCl add 76 ml to distilled water, use 1N HCL to adjust the pH to 1.2. 4 ℃ storage, can be stored for 2 weeks.
⑨ Solution B: Add 50 ml of 10 mM EDTA, 75 ml of 1M NaCl, 157.5 ml of 0.4M Na
2
HPO
4
solution B: add 50 ml of 10 mM EDTA, 75 ml of 1M NaCl, 157.5 ml of 0.4M Na 2 HPO 4, 92.5 ml of 0.2M citric acid to 120 ml of distilled water, and adjust the pH to 6.0 with 1N HCL or 1N NaOH. store at 4 ℃ for 1 month.
⑩ AO working solution: add 0.1 ml of AO stock solution to 9.9 ml of solution B. Prepare on ice on the day of use. EDTA improves the binding of AO to the RNA in the concentrated sample.
2.1 Instrument Use and Sampling
The excitation wavelength of AO ranges from 455 to 490 nm, and because the emission spectrum of DNA-bound AO overlaps with the red fluorescence from RNA, the combined use of filters can reduce this effect. For DNA, we recommend the use of 550-LP dichroic filters (long pass) in combination with 525BP (band pass), and for RNA, we recommend the use of 630-LP. Further adjustment of the compensation reduces the overlapping excitation light. A standard microsphere is required to monitor the status of the instrument during use to determine the consistency of the standard microsphere resolution with the detection channel peaks. The instrument records the scattered light signal, the DNA/RNA two-parameter histogram, and the parameters of each fluorescent signal distribution. Adherent cells must be identified.
2.2 RNA Content Criteria
RNA and DNA can be quantified by comparison with biological standards (lymphocytes, cell lines, etc.) that have the same contents. Freshly isolated lymphocytes are commonly used as a stable control. For experiments, both RNAase-treated and untreated controls should be set up. The cells to be tested must be measured under the same conditions as the control cells, and the RNA index is expressed as the ratio of the RNA content of the test sample to the RNA content of the control. Lymphocytes may be used as a DNA content criterion to determine the DNA index.
2.3 Criteria
Normal peripheral blood lymphocytes (NPBLs) can be used as a biological control for evaluating the quality of AO staining as well as diploid G0/G1, assaying channel stability. Lymphocytes are separated by Ficoll-Hypaqu gradient centrifugation and the cell concentration is adjusted to 1 × 106 cells/ml, which can be used to establish a range of acceptable peaks on the RNA detection channel. Each set of samples to be tested must be stained and manipulated at the same time as the control.
Caveat
1. Prepare storage solution prior to processing samples; AO working solution should be prepared at the time of sample staining.
2. All glassware used for reagent preparation must be free of RNAase.
3. the concentration and pH of the reagents must be configured exactly as required.
4. peripheral blood lymphocytes and bone marrow cells must be separated from solid tumors using Ficoll-Hypaque Gradient Separation Solution and bone marrow biopsies must be carefully pulverized to release individual cells.
5. Depending on the degree of blood cell contamination, fluids and needle aspirates are either washed directly with buffer or separated by gradient centrifugation.
6. Samples should be separated by applying a buffer containing MgCL2PBS containing MgCL 2 and the cell concentration is adjusted to 1 × 106.If membrane surface molecules are to be detected, it is not necessary to analyze the samples with MgCL2.2PBS containing MgCL 2 is not required for the detection of membrane surface molecules.
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