Operation method
RNA extraction and labeling
Materials and Instruments
Cells harvested from tissue culture Cells in tissue culture Frozen whole tissue Move See "Others" for details of "Materials", "Reagents" and "Consumables" required for the experiment. 1a. If starting from harvested tissue culture cells: Wash the cell sediment twice with PBS. 1b. If starting from tissue culture cells: add 1 ml of TRIzol per 2×107 cells and shake to mix. 1c. If starting from tissue: add 4 ml TRIzol per 100 mg of frozen tissue directly and homogenize with a rotary blade tissue homogenizer. 2. Add 1/5 v/v of chloroform. 2. Add 1/5 v/v of chloroform, shake for 15 s, allow to stand for 3 min. centrifuge at 12,000 g for 15 min at 4°C. 3. Gently transfer the supernatant to the tissue homogenizer. 3. Gently transfer the supernatant into a 15 ml polypropylene tube and note the volume. 4. 4. Add an equal volume of 70% ethanol (final concentration 35%) drop by drop while mixing. 5. 5. Add the supernatant from 2 x 107 to 1 x 108 cells to an RNeasy Maxi column in a 50 ml centrifuge tube. 6. 6. Centrifuge at 2880 g for 5 min at room temperature on the horizontal head of a clinical centrifuge. 7. 7. Collect the effluent and pour it back onto the top of the column, repeating Step 6 once. 8. Discard the effluent and add 15 ml of RWl to the column. centrifuge at 2880 g for 5 min at 20-25°C. 9. Discard the effluent and add 15 ml of RWl to the column. 9. Discard the effluent and add 10 ml RPE to the column. centrifuge at 2880 g for 5 min at 20-25°C. 10. Discard the effluent and add 10 ml RPE to the column. 10. Discard the effluent and add 10 ml of RPE to the column again. centrifuge at 2880 g for 10 min at 20-25°C. Discard the effluent. 11. Place the column in a new 50 ml conical polypropylene centrifuge tube and add 1 ml of DEPC-treated water (included in the kit). Allow to stand for 1 min. Centrifuge at 2880 g for 5 min at 20-25°C without discarding the effluent. 12. Add more DEPC-treated water to the column. 12. Add 1 ml of DEPC-treated water to the column. Place in the top tube and let stand for 1 min. centrifuge at 2880 g for 10 min at 20-25°C. 13. 13. Dispense the eluate into 1.5 ml microcentrifuge tubes at 400 ul each. 14. 14. Add 1/10 of a volume of 3 mol/L sodium acetate (pH 5.2) to each tube, then add 1 ml of 100% ethanol, shake to mix, and allow to stand at room temperature for 15 min. 15. 15. 4°C, centrifuge at 12,000 g for 15 min and wash the precipitate twice with 75% ethanol. The precipitate was washed twice with 75% ethanol and stored indefinitely in 75% ethanol at -80°C. 16. 16. Centrifuge at 12,000 g for 15 min at 4°C. Aspirate off the supernatant and allow to dry in air. Redissolve the RNA in DEPC-treated water to a concentration of approximately 1 mg/ml. 1 ul is added to 100 ul of 50 mmol/L NaOH and the A260 value is read to determine the concentration of RNA. 17. 17. Concentrate in Microcon YM-100 filter tubes by centrifugation at 500 g to a concentration >7 mg/ml and determine the concentration rate if necessary. 18. 18. Determine the concentration of the concentrated RNA sample spectrophotometrically. Store at -80°C. 19a. If using immobilized oligo-dT primers: Complex the primers with the RNA in the 17 ul reaction system below (use 0.2 ml thin-walled PCR tubes so that the incubation process can be performed on a thermal cycler): 19b. If oligo-d(T)12-18 primer is used: replate the primer with the RNA in the 17-ul reaction system below (use 0.2 ml thin-walled PCR tubes so that the incubation can be performed on a thermal cycler): 20. Heat at 65°C for 10 min and leave on ice for 2 min. 21. 21. Prepare a master mix (total volume 23 ul) containing the following components: 8 ul 5 x first chain buffer 4 ul 10× low T dNTP mixture 4 ul 1 mmol/L Cy5- or Cy3-labeled dUTP 4 ul 0.1 mol/L DTT 1 ul 30 U/ul RNasin 2 ul 200 U/ ul Superscript II 22. Add 23 ul of reaction mixture containing Cy5-dUTP or Cy3-dUTP to each tube. Blow and mix well, centrifuge quickly to concentrate the solution at the bottom of the tube. 23. 23. Incubate at 42°C for 30 min, then add 2 ul Superscript II to ensure that the enzyme is well mixed in the reaction system, and incubate at 42°C for 30-60 min. 24. Add 5 ul of 0.5 µg/kg of Cy5-dUTP or Cy3-dUTP to the reaction mixture. 24. Terminate the reaction by adding 5 ul 0.5 mol/L EDTA (pH 8.0). 25. 25. Add 10 ul 1 mol/L NaOH and incubate at 65 ℃ for 60 min to degrade the residual RNA. cool to room temperature. The purity of the sodium hydroxide used in this step is critical. Slight contamination or prolonged storage in glass containers can cause this solution to degrade Cy5 molecules and cause the solution to turn yellow. Some researchers have reduced the hydrolysis time to 30 min with better results. 26. Neutralize the solution by adding 25 ul of 1 mol/L Tris-Cl (pH 7.5). 27. 27. Place labeled cDNA and 400 TE buffer (pH 7.5) in a Microcon YM-100 column and mix by blowing. Blow and mix. 500 slices are used for 10 min at room temperature to desalinate. 28. Add 200 ul of TE buffer (pH 7.5) again and wash once, to approximately 20-30 ul (500 g for 8-10 min). 29. Invert the column into another clean collection tube and centrifuge at 500 g for 3 min at room temperature to recover the cDNA. In some cases, Cy5-labeled cDNA forms a gelatinous blue precipitate in the concentration tube. Seeing this means there is contamination. The more contamination there is, the higher the percentage of cDNA that will be adsorbed by the gel. Even if heated to dissolve, this stuff tends to bind non-specifically to the target DNA without recognition. 30. 30. Take 2-3 ul of Cy5-labeled cDNA for testing and the remaining 18-28 ul for hybridization. 31. 31. Run a 2% agarose gel with the labeled probe, using TAE buffer. To maximize the sensitivity of the fluorescence analysis of the gel, run the gel with the minimum amount of dye and do not add ethidium bromide to the gel or to the buffer. 32. Scan the gel with Molecular Dynamics' Storm Burst Scanner (parameters: red fluorescence, 20 um resolution, 1000V on PMT). See Figure 1 for a typical example of labeling success (lane A) and failure (lane B). Caveat All solutions need to be prepared with RNAase-free water (e.g., DEPC-treated water) unless otherwise indicated. Common Problems 1. Materials cells harvested from tissue culture, cells in tissue culture, frozen whole tissue 2. Reagents Phosphate buffer solution (PBS) TRIzol reagent (Life Technologies) Chloroform 100%, 75% and 70% ethanol Rneasy large scale kit (Qiagen) included: 50 ml large-scale centrifuge column and collection tube RW1 buffer RPE buffer DEPC-treated water (included in the kit) 3 mol/L sodium acetate, pH 5.2 2 mg/ml 固定化的 oligo-dT 引物（固定的：5’-TTT TTT TTT TTT TTT TTT TTV N－3’；如 Genosys 公司） 1 mg/ml d (T) 12-18 (Amersham Pharmacia Biotech) 10 x low T dNTP mixture 1 mmol/L Cy 3-dUTP or Cy 5-dUTP, store at -20°C, light sensitive. RNasin RNAase Inhibitor (Promega) Superscript II Reverse Transcriptase Kit, RNAase H-free, with 5× First Strand Buffer and 1 mol/L DTT (Life Technologies) 0.5 mol/L EDTA 1 mol/L NaOH 1 mol/L Tris-Cl, pH 7.5 TE buffer, pH 7.5 1 mg/ml human C0t-1 DNA (Life Technologies) 2% (m/V) agarose gel (6 cm wide x 8.5 cm long, 2 mm pore width) in TAE buffer 50 x TAE buffer 3. Consumables Tissue homogenizer (e.g. Polytron PT 1200 from Brinkmann Instruments) 15 ml round-bottomed polypropylene centrifuge tube 50 ml conical polypropylene centrifuge tubes Clinical centrifuge with horizontal head for centrifugation of 50 ml conical centrifuge tubes 1.5 ml microtubes Centrifuge filter tubes (e.g. Amicon Microcon YM-100) 0.2 ml thin-walled PCR tubes with lids Thermal cyclers Fluorescence scanner (e.g. Molecular Dynamics Storm system for gel analysis) For more product details, please visit Aladdin Scientific website.
Phosphate buffer (PBS) TRIzol reagent Ethanol Sodium ethanol EDTA NaOH Tris-Cl TE buffer Immobilized oligo-dT primers
Tissue homogenizer Thermocycler Fluorescence scanner
Figure 1 Fluorescence scan of Cy5-labeled cDNA after 2% agarose electrophoresis.