Combined application of flow cytometry and gene chip methods to detect transcriptional profiles
Combined application of flow cytometry and gene chip methods to detect transcriptional profiles
The two technologies, flow cytometry and gene expression profiling, share many common features: they involve complex, highly sophisticated equipment; they can provide methods to analyze cells that are unique and not easily accessible by conventional means; and they require extensive data analysis and archiving capabilities. However, they also differ in one particular, important concept: when cells are mixed together, flow cytometry (to which cell sorting techniques are similar) reduces the complexity of the cell mixture by analyzing the optical properties of the cells and separating them into homogeneous populations of cells, which are set aside for further study. Whole gene expression profiling techniques, on the other hand, provide information about all the genes within a particular biological specimen, and by their very complex way of obtaining the information, it can therefore only be simplified by means of data processing. If these two techniques are combined and specific labeling methods are devised depending on the cell type, it will be possible to isolate tissues, sort enough cells for subsequent analysis of full gene profile expression, and, in principle, it will be possible to characterize the pattern of expression of full gene profiles within any given type of cell.
Principle
The principle behind the combined application of flow cytometry and gene chip methods for transcriptional profiling is that by combining the two techniques, flow cytometry and gene expression profiling, and by devising specific labeling methods depending on the cell type, it is possible to isolate tissues, sort enough cells for subsequent analysis of full gene profile expression, and in principle, it is possible to identify the pattern of expression of full gene profiles within any given cell type.
Operation method
Combined application of flow cytometry and gene chip methods to detect transcriptional profiles
Principle
The principle behind the combined application of flow cytometry and gene chip methods for transcriptional profiling is that by combining the two techniques, flow cytometry and gene expression profiling, and by devising specific labeling methods depending on the cell type, it is possible to isolate tissues, sort enough cells for subsequent analysis of full gene profile expression, and in principle, it is possible to identify the pattern of expression of full gene profiles within any given cell type.
Materials and Instruments
Equipment: Move The basic process of the combined application of flow cytometry and gene chip methods to detect transcriptional profiles can be divided into the following steps: 1.1 Preparation of oligonucleotide spot samples 1.1.1 Add 15 μl of sterile distilled water (dH20) per well to a 384 microtiter plate containing 600 pmol of oligonucleotides. 1.1.2 In a Sorvall 5B centrifuge (Kendro Laboratory Products, Asheville, NC) equipped with a centrifugation basket for the 384 microplate, centrifuge the microplate rapidly at 200 g (1,000 rpm with a SH 3,000 rotor). 1.1.3 Transfer to a fixed-rail oscillator (e.g., Belly Dancer from Stovall Life Science, Greensboro, NC) and oscillate for 1 h at room temperature. 1.1.4 Resuspend the solution several times in the wells with a 12-channel micro-sampler and transfer 7.5 μl to a new 384 microplate. 1.1.5 Dry the microtiter plate in a SpeedVac (Savant Instruments, Holbrook, NY) at 50 °C. 1.1.6 Store one set of microplates at - 20 °C. Dispense the first set of microtiter plates (approximately 750 microdisplays from a 300 pm volume), followed by the second set. 1.2 Rehydration and spotting of oligonucleotide microarrays. 1.2.1 Add 8 μl of 3 × sodium citrate (SSC) per well. 1.2.2 Place on a fixed-track shaker at room temperature for 1 h. The oligonucleotides are now ready to be spotted. 1.2.3 The Arabidopsis Oligonucleotide Gene Chip is spotted onto an aminomethylsilane-impregnated SuperAmine slide using a TeleChem SMP3-type spotting needle. 1.2.4 The microarrays were spotted in units of 100 arrays (no recurring dots) with 160 μm spacing between the centers of each unit, using a 48-pin spotting head, 31% relative humidity, and re-dipping after every 50 slides. The speed of the counting head was set to a maximum of 10 mm/s. 1.2.5 Place at 80 °C for 1 h and dry the slides. Store at room temperature away from light. 1.2.6 Dry the microtiter plate with Speed Vac after counting. For subsequent spotting, we resuspended the samples with 10-(N/2) μl deionized water (diH20), where N is the number of times the array was spotted. 1.3 Fixed oligonucleotide array units. 1.1 Rehydrate the GeneChip slides, i.e., place them in water above 50 °C for 5~10 s; then immediately place them on a heating block at 65 °C for 5 s to dry. Repeat this step 3 or 4 times. 1.2 Crosslink the slides by UV, with the DNA oriented at 65 mJ/c㎡ by UV (e.g., Strata Crosslinker from Strata Genetics, La Jolla, CA) up to a maximum of 130 mJ/c㎡. 1.3 Wash slides in 1% sodium dodecyl sulfate solution (SDS) for 5 min at room temperature. 1.4 Soak the gene chips in 100% ethanol for 30 s and shake gently to remove SDS. 1.5 Centrifuge the slides in a Sorvall centrifuge at 200 g (1,000 rpm with SH 3,000 rotors) for 2 min and shake the slides dry. 1.6 Store the slides in a dry, light-proof box at room temperature.
① Gene chip plate: SuperAmine SMM (TeleChem International, Inc., Sunnyvale, CA);
② Gene chip spotting needle: SMP3 (TeleChem);
③ Gene chip dispenser: An OmniGrid 100 (GeneMachines, San Carlos, CA) equipped with Stealth dispensing head SPH48 (TeleChem);
④ 384-well plate (Genetix, New Milton, England, cat. no. X7001);
⑤ Oligo (dT)
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Dynabeads (Cat. No. 610.05) and 1.7 ml Magnetic Particle Concentrator (MPC) (Cat. No. 120.20) (both purchased from Oslo, Norway);
(vi) Nuclease-free 1.7 ml centrifuge tubes (Life Science Products Inc., Denver, Catalog No. 7 509-2 002);
(vii) Microcon column (Millipore, Billerica, MA, Catalog No. YM30);
⑧ Coverslips: 24 mm × 60 mm, Hybri-Slips (Sigma, St. Louis, MO, catalog number H-0 784).
Reagents:
① Binding buffer: 1 M LiCl, 2 mM EDTA, 20 mMTris-HCI, pH 7.5, made up to a total volume of 50 ml with RNAase-free water;
② Wash buffer: 0.15 mM LiCl, 10 mM Tris-HCl, pH 7.5, made up to a total volume of 50 ml with RNAase-free water;
③ Elution buffer: 10 mM Tris-HCl, pH 7.5, made up to a total volume of 15 ml with RNAase-free water;
④ Regeneration buffer: 0.1 M NaOH;
⑤ Storage buffer: 250 mM Tris-HCI, pH 7.5, 20 mM EDTA, 0.1% Tween-20 and 0.02% azide Na;
(vi) Liquid blocking agent (Amersham Biosciences, Piscataway, NJ, catalog number RPN3 601);
⑦ Cy3- and Cy5-dUTP (Amersham Biosciences, cat. no. PA53 022 and PA55 022);
⑧ PowerScript reverse transcriptase (BD Biosciences Clontech, Palo Alto, CA, catalog nos. 8460-1, 8 460-2);
⑨ Trizol (Invitrogen, Carlsbad, CA, Catalog No. 15596-026);
⑩ RNAase inhibitor (Invitrogen, catalog no. 15 518-0 120).
Primers: random 15-mer primers (Qiagen-Operon, Catalog Nos. SP180-1, SP180-2); random hexamer primers (Invitrogen, Catalog No. 48-190-011)
2.1 Preparation of RNA
2.1.1 Preparation of total RNA
(1) Wear gloves! RNA is particularly unstable and easily contaminated due to the prevalence of ribonucleases. All aqueous solutions should be prepared in deionized water treated with diethylpyrocarbonate (DEPC). Glass instruments should be autoclaved.
(2) Freeze the mortar, including the rods, by adding approximately 100 ml of liquid nitrogen. Add 200 mg of tissue when the liquid nitrogen is half evaporated.
(3) Crush the tissue rapidly with the rod, taking care to avoid losing the sample. When the liquid nitrogen has completely evaporated, increase the rate of grinding until a fine talc-like powder appears.
(4) Add 1 ml Trizol for every 100 mg of tissue and continue to grind with the rod. If the liquid mixture freezes, wait a little to allow it to melt slightly, then continue grinding.
(5) When completely mixed, allow the slurry to thaw (cover the mortar with aluminum foil while waiting) and transfer 1 ml of slurry to a 1.5 ml RNAase-free tube using an Eppendorf pipette.
(6) Incubate for 5 min at room temperature.
(7) Add 0.2 ml of chloroform per 1 ml of Trizol volume and mix for 15 s.
(8) Incubate for 1 min at room temperature.
(9) Centrifuge at 15 300 g for 10 min at 4 °C.
(10) Transfer the supernatant to a new RNAase-free tube and place immediately on ice. During transfer, remove the aqueous phase from the top and edge of the tube with a pipette, leaving the buffer strip above the bottom (organic) layer intact.
(11) Add an equal amount of isopropyl alcohol to precipitate. Invert the tube twice, mix well and incubate on ice for 15~30 min.
(12) After centrifugation at 15,000 g for 10 min at 4 °C, a small white flaky precipitate is visible (the size of the precipitate depends on the amount of raw material, but we used 200 mg of tissue and should see a precipitate). Occasionally, the precipitate may be purple or brown, depending on the type of raw tissue used or the conditions under which the plant was grown.
(13) Decant the supernatant. Centrifuge rapidly in a microcentrifuge for 1 min and discard the residual supernatant with a 200 μl Eppendorf pipette.
(14) Wash the sediment with 1.0 ml of 75% ethanol in RNAase-free (DEPC-treated) water, being careful as the sediment may become loose. Discard most of the ethanol with a pipette.
(15) Invert the tube onto Kimwipe absorbent paper and air-dry the precipitate for 5 min at room temperature (do not dry too long as the precipitate will be difficult to resuspend).
(16) Add 25 μl of RNAase-free water and resuspend with a 200 μl Eppendorf pipette (break up the precipitate to help it dissolve). Incubate on ice for at least 1 h, occasionally resuspending with a pipette. Characterization of the RNA is rapidly assayed on a 1% agarose gel prepared in TBE (Tris-borate-EDTA) buffer (see Ref. 46 for details of this routine).
(17) Solubilized RNA was centrifuged at 20 000 g at 4 °C for 15 min.
(18) Transfer the supernatant to a new RNAase-free tube. Note: The supernatant and the layer of unwanted debris below it are not always clearly distinguishable.
(19) (Optional) Repeat precipitation is necessary only for "difficult" tissues (3 times for corn endosperm, 1 time for root tissue). Repeat the precipitation with 10% volume of 3M sodium acetate and an equal volume of 100% isopropanol. Place precipitates on ice for 1 h (or -80 °C overnight).
(20) Centrifuge at 20,000 g for 20 min at 4 °C.
(21) Place on ice, dissolve in 25 μl of RNAase-free water for 1 h, and resuspend.
(22) Centrifuge at 20 000 g for 20 min at 4 ℃.
(23) Determine the RNA concentration spectrophotometrically. If the concentration is <100 ng/μl, repeat the isopropanol precipitation.
(24) Run electrophoresis on a 1% agarose gel prepared in 1 × TBE (Tris-borate-EDTA) buffer to detect the integrity of RNA.
(25) Store the remaining RNA at -80 °C.
2.1.2 Preparation of poly A+ RNA from total RNA using Dynabeads
(1) Shake the Dynabeads well (Dynabeads are brown) and transfer 200 μl (2 times the volume of total RNA) of the suspension into a nuclease-free 1.7 ml tube.
(2) Place the tube on the Dynal MPC magnet for 1 min. check that the beads and solution are clearly separated, and discard the storage buffer from the tube with a pipette without destroying the precipitate.
(3) Remove the tube from the MPC and resuspend the beads in 200 μl of binding buffer and mix with a pipette.
(4) Repeat steps 2 and 3 and the Dynabeads are now ready to use.
(5) Transfer 75 μg of total RNA with 100 μl of DEPC-treated sterile water to a nuclease-free 1.7 ml test tube and add 100 μl of 2 × Binding Buffer.
(6) Incubate the RNA at 65 ℃ for 2 min, and then add the binding buffer/Dynabeads suspension.
(7) Mix well with a pipette, gently invert the tube and anneal for 3~5 min at room temperature.
(8) Place the tube on the MPC magnet for 30 s or until the suspension is clear, and discard the supernatant with an Eppendorf pipette.
(9) Remove the tube from the MPC magnet and wash the beads twice with 200 μl of wash buffer, concentrating the beads with the MPC magnet each time before discarding the supernatant.
(10) Add 10 μl of elution buffer and incubate for 2 min at 80 ℃ to elute the mRNA from the Dynabeads. immediately place the tube on the MPC magnet and quickly transfer the supernatant to a sterile 1.5 ml test tube without destroying the precipitate. If the eluted mRNA is not to be used immediately, store at -70 °C.
(11) pH 8.0 TE (Tris-EDTA) was used as buffer for quantitative determination of RNA by spectrophotometer at 260/280 nm. a ratio of A260/A280 of 1.8~2.0 indicated that the prepared poly(A)+ RNA was pure. the yield of poly(A) + RNA should be 2%~3% of the total RNA.
(12) Dynabeads can be used up to 4 times, add 150 μl of Reconstruction Buffer to regenerate Dynabeads, and mix well with a pipette.
(13) Incubate the tubes at 65 ℃ for 2 min.
(14) Place the tube on the MPC magnet and discard the liquid when clarified.
(15) Remove the test tube from the MPC magnet, add 200 μl of Reconstruction Buffer and mix well with a pipette.
(16) Place the test tube on the MPC magnet and discard all liquid.
(17) Remove the test tube and add 200 μl of Storage Buffer.
(18) Place the tube on the MPC magnet and discard the buffer when clarified.
(19) The Dynabeads can now be reused, starting with step 5.
2.2 Labeling RNA for oligonucleotide gene chip hybridization
2.2.1 Fluorescent target product from total RNA
(1) Label two 0.5 ml test tubes with "Cy3" and "Cy5" and cover the tubes with aluminum foil to protect them from light (the dye is light sensitive). Hybridize with a pair of Cy3- and Cy5-labeled RNA target sequences, which are prepared separately and mixed before hybridization. Always keep RNA on ice unless otherwise indicated.
(2) Mix the following in 2 0.5 ml tubes: 20-50 μg of RNA until 20.0 μl, 2.0 μl dNTP (10 mM dATP, dCTP, dGTP, 2 mM dTTP), 2.0 μl Cy3 or Cy5-dUTP (1 mM), and 2.0 μl oligo-dT primer (0.5 μg/μl).
(3) Incubate the mixture at 65 ℃ for 5 min.
(4) Add: 8.0 μl of 5 × first strand buffer, 4.0 μl of 0.1 M DTT, 1.0 μl of RNAase inhibitor (10 U/μl), and 1.0 μl of PowerScript RT, and replenish to 40.0 μl to heat the mixture.
(5) Incubate the tubes at 42 ℃ for 2 h. This step is easy to perform by programming the PCR instrument.
(6) Add 5 μl of 0.5 M EDTA and 5 μl of 1 N NaOH and incubate at 65 ℃ for 10 min.
(7) Add 25 μl of 1 M Tris-HCl pH 8.0 and 100 μl TE. Store on ice or refrigerate at -20 °C overnight until ready for purification. Ensure that the tubes are completely wrapped in aluminum foil.
2.2.2 Fluorescent target product from poly( A+ ) RNA
(1) Label two 0.5 ml test tubes with "Cy3" and "Cy5" and cover the tubes with aluminum foil to protect them from light (the dye is light sensitive). Perform each hybridization with a pair of Cy3- and Cy5-labeled RNA targets; the sequences of the two labeled RNA targets are prepared separately and mixed before hybridization. Always keep RNA on ice unless otherwise indicated.
(2) Mix the following in each 0.5 ml tube:
1 to 2 μg poly(A) + RNA until 20.0 μl, 2.0 μl dNTP (10 mM dATP, dCTP, dGTP, 2 mM dTTP), 2.0 μl 1 mM Cy3 or Cy5-dUTP, and 1.0 μl 3 μg/μl of randomized hexamer primer.
(3) Incubate the mixture at 65 ℃ for 5 min and freeze on ice for 5 min.
(4) Add: 8.0 μl 5 × first strand buffer, 4.0 μl 0.1 M DTT, 1.0 μl RNAase inhibitor (10 U/μl), and 2.0 μl PowerScript RT, replenished to 40.0 μl, to heat the mixture.
(5) Incubate the tubes at room temperature for 20 min, and then incubate the tubes at 42 ℃ for 2 h (this step should be operated by PCR instrument).
(6) Add 5 μl of 0.5 M EDTA and 5 μl of 1 N NaOH and incubate at 65 ℃ for 10 min.
(7) Add 25 μl of 1 M Tris-HCI pH 8.0 and 100 μl TE. Store on ice or refrigerate at -20 °C overnight until ready for purification. Ensure that the tubes are completely wrapped in aluminum foil.
2.3 Target Sequence Purification
Purification of target sequences is based on volumetric exclusion using nitrocellulose filter membranes in Microcon YM30 columns. These membranes block single-stranded DNA molecules larger than 55 bp and other molecules with molar molecular weights >30,000.
2.1 Note the labeling of each pair of columns and tubes and their corresponding target sequences ready for purification and labeled.
2.2 Transfer the labeled target sequence to the centrifuge column using a 200 μl Eppendorf pipette without touching the membrane.
2.3 Centrifuge at 15 °C, 11,700 g for 15 min (ensure that no liquid remains in the column).
2.4 Add 100 μl of TE to each column and mix the solution with a pipette, being careful not to damage the membrane. Prevent the solution from flowing out of the column to prevent membrane rupture.
2.5 Centrifuge the columns at 15 °C, 11,700 g for 15 min.
2.6 Repeat the TE wash (steps 4-6) a total of 4 times.
2.7 After 4 washes, add 40 μl of TE to the column and centrifuge. Mix carefully with an Eppendorf pipette.
2.8 Leave at room temperature for 2 min.
2.9 Upside down the column and place in a new centrifuge tube and centrifuge for 1 min at 11 700 g at 15 °C.
2.10 Immediately hybridize with labeled target sequences, or cover with aluminum foil and store in the refrigerator at -20 °C.
2.4 Gene chip hybridization
Hybridize with paired Cy3- and Cy5-labeled target sequences.
2.4.1 Mix the following reagents in a centrifuge tube: 25.0 μl 20 × SSC, 16.0 μl Liquid Blocker, 10.0 μl 2% SDS, X μl labeled target, and diH2O to 250 μl.
2.4.2 Denature the labeled target sequence by placing the tube in boiling water for 2 min. Quickly place the tube on ice.
2.4.3 Place the GeneChip slide (DNA side up) on a heating block at 65 °C, add the labeled target sequence dropwise and cover with 24 mm × 60 mm Hybri-Slip plastic to avoid air bubbles. A raised coverslip is recommended.
2.4.4 Incubate slides in a humidified chamber at 65 °C for 8 to 12 h. A simple humidified chamber can be constructed from disposable Petri dishes as follows:
(1) Place the bottom portion of a 100 mm × 15 mm square Petri dish upside down inside a large, round 150 mm × 15 mm Petri dish (VWR International, catalog no. 25 384-139).
(2) Add 5 ml of distilled water to the round Petri dish.
(3) Place the whole Petri dish in a 65℃ oven and equilibrate for 1~2 hours.
(4) Place the gene chip slide on top of the square Petri dish and put the lid on the round Petri dish. An example of this method is available at www.ag.arizona.edu/microarray.
2.4.5 Wash the slides at 55 °C for 5 min in 2 × SSC + 0.5% SDS, 0.5 × SSC for 5 min at room temperature, and 0.05 × SSC for 5 min at room temperature.
2.4.6 Drain the slides in a Sorvall centrifuge at 200 g (1 000 rpm in a Sorvall SH 3 000 rotor). The slides were immediately scanned with a GeneChip Scan Recorder according to the manufacturer's recommendations.
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