Toxicological effects of environmental toxins detected by cDNA macroarrays and gene expression profiles
Toxicological effects of environmental toxins detected by cDNA macroarrays and gene expression profiles
This chapter describes how to prepare and use cDNA macroarrays to detect the effects of environmental toxins on gene expression, and describes the materials and methods used in the experiments. Since commercial microarrays are not commercially available for some non-traditional species, the methods described in this chapter allow researchers to design and use their own microarrays for their own experimental purposes.
We have intentionally omitted statistical data from our experiments. We have intentionally omitted the content of statistical analysis in the experiments because the methods of statistical analysis still need to be further developed, and different experiments must be targeted to apply different statistical methods. In conclusion, gene macroarrays are relatively simple and high-throughput assays.
Written by Martin, this experiment is from "Environmental Genomics Experiment Guide".
Operation method
Detection of toxicological effects of environmental toxins by cDNA macroarrays and gene expression profiles Move I. MATERIALS 1. amplification and preparation of clones (1) Glycerol-preserved E.coli, plasmid containing the target cDNA insert. (2) Tag polymerase and buffer (New Englang Biolabs cat. no. M0267). (3) 10 mmol/L dNTP mixture. (4) The primers used to amplify the inserted fragments in the vector were diluted to 10 μmol/L (M13, SP6, T4 or T7 were commonly used). (5) 70% ethanol in nuclease-free water. (6) Tris-EDTA buffer: 10 mmol/L T ris, lmmol/L E D TA , pH 8.0. (7) Ordinary 96-well round-bottom culture plate. (8) 96-well plate culture sealer (breathe easy strips, USA Scientific cat. no. 9123-6100). (9) L B medium: l0 g tryptone, 5 g yeast extract, 10 g NaCl, pH 7.0 per liter. (10) Milipore filtration system for purification of PCR products (MultiScreen Filtration System Vacuum Manifold cat. no. MAVM0960R and Montage PCR96 Cleanup plates cat. no. MAVM0960R). MANU03010). (11) 96-well PCR plate for PCR instrument. (12) Multi-channel pipettes (20 μL and 200 μL). (13) Plain flat-bottomed 96-well or 384-well plates. (14) Agarose gel electrophoresis system for PCR product electrophoresis. (15) Quality standards for DNA molecules between 300 and 2000 bp in size. (16) UV spectrophotometer, preferably with plate reader. (17) A 96-well plate for use with the UV spectrophotometer (refer to instrument description). (1) Pre-cut Pall Biodyne B neutral nylon membrane (N unc, cat. no. 250385). (2) Spot Report Array Validation System (Stratagene, cat no. 252005-7), including controls [poly (dA), human Cot-1, Arabidopsis cDNA, empty vector, etc.], which also need to be dot-printed on the array. (3) UV transilluminator (we used a UV Stratalinker 1800, Stratagene). (4) Centrifuge with 96-well plate rotor (any model will do). (5) Autosampler (e.g. Biomek 2000 with IOOnL needle, Beckman Coulter). (6) Distilled water. (7) 1 0 % bleach. (8) 70 % ethanol. (9) Bromophenol blue. (1) M-MuLV reverse transcriptase and buffer [New England Biolabs, cat. no. M0253S (200 U/mL)]. (2) Random hexamer primers (NewEnglandBiolabs, cat. no. S1254S) were added to 8 0 μL of nucleic acid-free enzyme-free sterile water to give a final concentration of I A26. units. (3) 10XdNTP mixture: can be purchased as a finished product or made by mixing 20 μL each of 100 mmol/L dCTP, dGTP, and dTTP with 10 μL of 100 mmol/L dATP. Add nuclease-free sterile water to a total volume of 400 μL (final concentrations of dCTP, dGTP, dTTP 4 mmol/L and dATP 2 mmol/L). (4) [ α-33P ] dATP: Available for purchase (Perkin Elmer cat. no. NEG612 H for 250 μCi). (5) Spike R N A mixtures: kits with the spike controls needed to prepare the arrays can be purchased from Stratagene, or spike RNA can be purchased separately (Spike 2 R C A and Spike 3 rbcL, cat. no. 252202 and 252203). (6) 20XSSC: 3 mol/L NaCl, 0 -3 mol/L sodium catalase, pH 7. 6, prepared in nuclease-free sterile water. Autoclave and leave at room temperature. (7) 2 0 % SDS: 200 g sodium dodecyl sulfate in I L of sterile water. (8) Hybridization buffer: 0.375 mol/L NaCl, 0.0375 mol/L sodium citrate, 7 % SDS and 25 % freshly prepared formamide in nuclease-free sterile water to 500 mL. add 100 m g of yeast tRNA. Store at 4 °C. (9) Wash 1: 2 XSSC-0.5% SDS (100 mL 20XSSC, 25 mL 20% SDS and 875 mL water). (10) Wash 2: 0.5 XSSC-0.5 % SDS (25 mL 20XSSC, 25 mL 20 % SDS and 950 mL water). (11) 10 mmol/L EDTA, pH 8. 0. (12) A kit to remove excess nucleosides from the labeling reaction (e.g., Qiagen QIAquick Nucleotide RemovalKit, cat. no. 28304). (13) Constant temperature metal bath. (14) Liquid scintillation counter and buffer. (15) Hybridization bottle (Fisher Scientific). (16) Hybridization oven (we used Labnet's MaxiOven). (1) Transparencies: available at any office supply store. (2) Scaled light screen: Molecular Devices. (3) Mercy Light Imaging System: We used Molecular Devices Typhoon Scanner. (4) ImageQuant software: Molecular Devices. (1) Add 98 μL of LB liquid medium containing the appropriate concentration of antibiotics to each well of a 9 6-well plate. (2) Add 2 glycerol-preserved bacterial fluids with the target plasmid to the LB medium (1 clone per well). An empty vector should also be included as an additional control for the array (the hybridization signal should be very weak when hybridizing to the target sample). (3) Cover the plate with a breathe easy strip and put the lid on the plate. (4) Incubate overnight at 37°C with shaking (approx. 125 r/min). (5) The following morning, prepare premixed PCR reaction solution (Table 1) at 90 per reaction (if performing whole-plate PCR, prepare sufficient mixture for 100 reactions). The experimental flow is shown in Fig. 1A. (6) Add 90 of the mix and 10 of the overnight culture to each well. (7) Cover the PCR plate with a suitable lid and cycle the reaction as described in Table 1. (8) At the end of the reaction, the PCR products were detected with 1 % agarose gel to confirm the amplification results. 5 μL of each reaction product was sampled together with the DNA molecular quality standard. Clones that fail to amplify are subjected to a new PCR reaction. (9) When all clones have been amplified, sample the reaction products through a Millipore filtration system, allowing the liquid to flow through the filter membrane. Wash once with 70% ethanol. (10) Turn off the vacuum pump and resuspend the PCR products in 50 T buffer. Allow the products to stand at room temperature for a few minutes before transferring them to another 96-well plate (a multichannel pipette can be used). (11) Dilute 5 µL of purified DNA into 95 µL of water and measure the concentration using a UV spectrophotometer. (12) If the amount of product is not enough you can re-amplify the clone and the re-amplified product can be combined with the first one. The concentration required for array dot blotting should not be less than 160 ng/ V L . (1) Before starting this step, you should first arrange the position of the genes to be dotted on the array. We tend to spot genes in duplicates (see Note 1), not forgetting that control spots must be included in the array (see Notes 2 and 3). (2) Dilute the purified DNA (3000 ngDNA in 18.75 μL of water to a final concentration of 160 ng/μL) into the corresponding wells of the plate to be used for dot blotting. (3) Add 1.9 μm 3 mol/L NaOH to each well. (4) Cover the plate and incubate at 65°C for 15 min (we used a hybridization oven). (5) Immediately place on ice and let stand for 2~3 min. (6) Centrifuge briefly. (7) Remove the plate cover and add 9.35 μL of 20XSSC containing bromophenol blue to each well and blow to mix (the volume should now be approximately 30 μL). (8) Centrifuge again briefly at 100 g for 5 s (this step is important). (9) Load the sample plate, nylon membrane, bleach, water and ethanol into the autosampler and start counting. The program should be pre-written for the experiment prior to counting (see Note 4). . (10) Before counting another set of samples (when all arrays on the bench have been counted), the counting needle must be cleaned. Program the instrument to submerge the needle in bleach, water, and ethanol for 10 s, and fan-dry it before counting. A new set of membranes can be prepared while the needle is being cleaned. (11) Crosslink the DNA with a UV transilluminator to immobilize it on the membrane. The irradiation dose is the same as for Southern blotting (100 mJ). (12) More than 100 membranes can be spotted with high precision using a 100 nL spotting needle. (1) Remove radioactivity from the ultra-low temperature refrigerator and place behind a Plexiglas shield. Turn on the 37°C and 100°C water baths. (2) Turn on the hybridization oven, set the temperature at 64°C and preheat the hybridization buffer in the oven. (3) In a microcentrifuge tube, add 2 μL of random primers, 0 -65 μL of spike RNA mixture and 2 μg of sample RNA, and add DEPC water to a reaction volume of 1 3 μL. (4) Place the tube at 64°C for 5 min, then cool at room temperature for 5-10 min to anneal the primers. (5) After the centrifuge tube is cooled, add 2 μL of 10X RT buffer, 2 μl of 10X dNTP mix, 1 μL of MM uLV and 2 μL of [ α-33P ] dATP (20 μL in the centrifuge tube). (6) Incubate at 37°C for 1.5 to 2 h. Incubate at 37°C for 1.5 to 2 h. (7) Centrifuge the liquid to the bottom of the tube and then open Centrifuge 1. () Purify the labeled sample with a denucleotide kit. () A portion of the sample (we took 2 labeled samples) was taken to measure the intensity of the radiation using a liquid scintillation counter. (1) Begin prehybridization of the membrane. Place the membrane in a hybridization vial, add 5 to 6 mL of hybridization buffer (see Note 5), return the vial to the hybridization oven, and turn it over (12 to 14 r/min) at 64°C for 1.5 to 2 h (Fig. 2A). (2) Calculate the volume of radiolabeled cDNA required for hybridization (Equation 1). Each membrane needs to be hybridized at the same radiation intensity (l X 106cpm/mL ). Formula 1: Calculation of the amount of labeled cDNA Probe volume = 1 000 000 cpm/mundefined V /cpm/μL The probe volume is the volume of labeled cDNA in μL, and V is the volume of hybridization buffer in mL. 10 mmol/L EDTA, 20 times the volume of the probe volume calculated above, is then added to the probe volume. Then 10 mmol/L EDTA, which is 20 times the volume of the probe calculated above, was added (Equation 2). Equation 2: Calculation of the amount of ED TA to be added Probe volume 20_~L_μL 10 mmol/L EDTA added to the probe (3) Add the volume of cDNA calculated using Equation 1 to the 10 mmol/L EDTA calculated using Equation 2. Mix the two in a 1.5 m L centrifuge tube and snap or screw the cap on tightly to prevent the cap from loosening during denaturation. (4) Denature the probe at 100°C for 5 min, then place on ice for 2 min. (5) After centrifugation at 2700 g for 1 min, the solution was pipetted out and transferred to the corresponding hybridization flask, in which the membrane and hybridization solution had been placed. (6) Gently shake the hybridization flask, and then return it to the hybridization oven and rotate it at 64°C overnight for 14 h at about 12~15 r/min. (7) Place Wash 1 and 2 in the hybridization oven at 64°C overnight. There should be enough space on the inside edge of the hybridization oven for the Wash. (8) The next morning the membrane can be washed. The washing process is as follows (Figure 2B). a. First, dispose of the radioactive waste by pouring out the hybridization solution according to the requirements of the research institution. Add Wash 1 to each hybridization vial, making sure that the wash solution covers more than half of the membrane. Return to the hybridization oven and rotate at 12~14 r/min for 30 min. b. Wash 3 times with Wash 1 and then 4 times with Wash 2 (also for 30 min each time). Collect all waste liquids for safe disposal as required. c. The process should end with a total of 8 membrane washes. (9) Remove the film from the bottle, let it dry, and place it in the synchrotron dark box (see Note 6). (Note: Place the transparency in the raster area of the dark box and line up the film with the raster in a straight line so that the film can be easily analyzed, see Note 7). After 48 h of development on a matte screen, the film is scanned with a typhoon scanner. After exposure, the membrane was removed from the dark box and the pituitary screen was taken to scan the signal. We used the ImageQuant 5.1 software package (Molecular Devices) to quantify the sample points. The images were first adjusted for contrast (just visually, it won't affect later analysis), and then pulled up two (one around each point) around the largest or strongest pair of points (one pair of points for a gene). All other genes are selected with the same size box copied over, so that all genes are guaranteed to have the same size background area. (1) Once all the genes are boxed and all the data on all the membranes have been collected, calculate the mean value of the signal for each gene pair and subtract the mean value of all the blank spots on the array. (2) The genes on each membrane are then homogenized so that the signals are comparable from membrane to membrane. This step may entail adjusting each membrane against a specific mean or median, usually the membrane with the strongest signal. (3) The data are then log-transformed to better fit a normal distribution. An expert in statistics or bioinformatics can be consulted. (4) Use appropriate statistical models and methods to compare the differences you want to detect before the experiment begins. (5) Once the target gene is screened, it needs to be verified by other methods, such as quantitative PCR. The most important thing to consider before starting a gene array experiment is how the final data will be analyzed. This includes the number of individuals needed for each set of treatments, whether different individuals will be combined, what comparisons will be made and the statistical methods that will be used to make these comparisons. Since array analysis methods are still evolving, it is best to consult with statisticians and bioinformaticians before the experiment begins. Similarly, there are many ways of elaborating the data, all depending on the type of experiment. Caveat 1. We generally spot the genes two-by-two repetitively, which prevents possible instrumental spotting errors. 2. When lining up the genes on the array, it is important to remember to put a control on it. The controls we use are an empty vector amplification product, a few DNA-free spots (just 20XSSC with Bromophenol Blue), mRNA for ribosomal proteins, a few Stratagene array spot reporter kits for spikes and other cDNAs. 3. Since array spot printing is costly, it is possible to control the cost of the experiment by rationally designing two or more arrays to be printed on a single membrane. 4. Nowadays, manual dot blotters can be purchased, so there is no need to use an automated dot blotter, but when dot blotting a large number of arrays, a manual instrument will be more laborious and less reproducible. Vacuum-driven dot transfer machines may also be used for dotting, but this method has not been tried and may be time-consuming. 5. We generally use 5 m L of hybridization buffer, but the exact volume used depends on the volume of the hybridization vial. The larger the bottle, the more buffer is needed if half of the membrane is to be immersed in the liquid. 6. If a phosphor imaging system is not available, ordinary X-ray negatives can be used, but the dynamic range of these negatives is several orders of magnitude less than that of phosphor screens, so differential analysis is relatively difficult. If a negative is used, optical density analysis can be used in place of ImageQuant or other poor photographic imaging software.7 Scan the film asymmetrically on a phosphor screen so that the film corresponding to the image can be easily distinguished when the image is scanned. For more product details, please visit Aladdin Scientific website.
(10) 20XSSC: 3 mol/L sodium chloride, 0.3 mol/L sodium citrate, pH 7.6, prepared in nuclease-free sterile water. Autoclaved to room temperature. 0.01 mmol/L bromophenol blue was added after sterilization.