Comparative Molecular Physiological Genomics - Heterozygous hybridization experiments with cDNA arrays

(1) Add diethyl ether pyrocarbonate (DEPC) (Sigma-Aldrich, St. Louis, MO) to water at a final concentration of 0.1 %. 1 % and stirred overnight (>12 h), autoclaved. Pipettes, tubes, and other plastic or glass
Pipettes, tubes, and other plastic or glass products can be purchased RNase-free or stirred in DEPC water overnight to remove the RNase.This part of the experiment requires the use of DEPC water and RNase-free plastic or glass products for all of the solution preparation and dissolution of the RNA samples.

(2) TRIzol solution (Invitrogen, Carlsbad, CA).

(3) Chloroform (Fisher Scientific, Fairlawn, NJ).

(4) Isopropyl alcohol (Fisher Scientific).

(5) 70% ethanol. Add 30 mLDEPC water to 70 mL of anhydrous ethanol (Pharmco, Brookfield, CT).

(1) Oligotexpoly (A )+ mRNA Extraction Kit (Qiagen). NOTE: The 3 buffers listed below are kit companion reagents. , , , , , , , , and

(2) Oligotex Binding Buffer (OBB): 20 mmol/L Tris-HCl, pH 7.5, lmol/L NaCl, 2 mmol/L EDTA, 0.2% (W V ) decahydrate. 2 % (WV) sodium dodecyl sulfonate (SDS; Sigma-Aldrich).

(3) Oligotex Wash Buffer (OWB): 10 mmol,/L Tris-HCl, pH 7.5, 150 mmol/L NaCl, I mmol/L EDTA.

(4) Oligotex Extraction Buffer (OEB): 5 mmol/L Tris-HCl, pH 7. 5.

(1) 1μg mRNA sample.

(2) Polymerase chain reaction (PCR) instrument [e.g., Bio--Rad iCycler (Bio--Rad), PTC-100 (MJ Research)].

(1) Church's buffer: 0.25 mol/L _Na2HPO4, 0.25 mol/L _NaH2PO4, pH 7.5, 7 % SDS (W/V)

(2) 20XSSC: 3. 0 mol/L NaCl, 0-3 mol/L sodium catalase (Sigma-Aldrich).

(3) 2 0 % SDS (W / V )

(4) Yeast tRNA (10 mg/mL) (Invitrogen).

(5) Calf thymus DNA (10 mg/mL) (Sigma).

(6) DIG Easy Hybe solution (Roche).

(1) X-ray negative or phosphor screen imaging system (for ^ATLASTMcDNA arrays).

(2) Microarray Analyzer (to analyze human 19K cDNA arrays). There are many companies (AlphaInn tech, Affymetrix, VersArrayChipReader) that sell array analyzers, but there are some companies or services (and many institute facility centers) that can provide array scanning and analysis services for a fee, which is much more economical than purchasing an analyzer.

3 . Downloadable analysis software (e.g., Scanalyze; http://rana.lbl.gov).

(1) DNAman software (Lynnon Biosoft).

(2) Elicitation Design Software (Scientific and Educational Software).

(3) BicrRadiCycler (Bio^Rad) or other gradient PCR instrument.

(4) 50XTAE buffer: 242 g Tris base, pH 8. 5, 57. Im L glacial acetic acid, 37.2 g EDTA, IL distilled water.

(5) 1 % TACE agarose gel: IX TACE buffer, 1 % agarose (W/V), brominated ethyl ingot (1 μg/mL) added to 100 mL of water.

(6) DNA Sampling Dye: 0.25% (W/V) bromophenol blue, 0.25% (W/V) xylene cyan, 5 0% (W/V) glycerol.

(7) DNA molecular quality standards (Invitrogen). Select the appropriate molecular mass standard (from 100 bp to several kilobases) for the size of the desired PCR product.

Before starting any microarray experiment, it is important to select a suitable control and time point so that the data obtained are biologically meaningful. See Notes 1 and 2 for information on how to select suitable controls.

(1) Prepare a 1 % agarose-formaldehyde denaturing gel, submerged in IXM OPS buffer, with the wells of the gel completely covered by the solution. Pre-electrophoresis the gel for 15 min (while preparing the RNA sample).

(2) Take an appropriate volume of total RNA (containing 10 to 20 μg of RNA), add it to a labeled tube on ice, and adjust the total volume to 15 μL with DEPC water. 15 μL of RNA Sample Buffer and 6 μL of 6X RNA Sampling Buffer are added to each tube.

(3) Incubate the samples at 55°C for 10 min, and quickly place on ice. Add the appropriate volume of RNA Sampling Buffer to each tube to achieve a final concentration of I X Sampling Buffer in each sample.

(4) Gently mix the RNA samples, centrifuge briefly, and collect all samples at the bottom of an Eppendorf tube.

(5) Add all samples from each tube to the gel wells and record the order of addition.

(6) Perform electrophoresis at 100 V. End electrophoresis when the tip of the indicator dye reaches the bottom of the gel. Place the gel on a plastic film and observe the results under a UV light. The 28S and 18S ribosomal RNA (rRNA) bands are used to judge the quality of the RNA, and the ratio of the two should be about 2:1. This step ensures the quality of total RNA prior to mRNA extraction for DNA array hybridization. Although total RNA can also be used for probe synthesis, we recommend the use of mRNA. RNA quality is considered to be poorer when the ratio of 28S rRNA to 18S rRNA bands is much less than 2:1 or when the sample bands are diffuse (see Note 3 for the maximum amount of RNA used for microarray analysis).

(1) Preheat the PCR instrument to 70°C and add Iμg (at least 0.5 (ug/μL)) of each mRNA sample (control or experimental) to each 0. 5 m L PCR tube (or 0 -2 m L tubes depending on the size of the wells on the PCR instrument's heating module). Add 100 ng of 01igo-5' _-dT20N-3 ' and 100 ng of random primers to each tube (200 ng total) to ensure complete labeling of the entire mRNA library.

After drying, each was dissolved in 5 / μL water or TE (10 mmol/L Tris minus , pH 8.0, I mmol/L EDT A ) buffer, and the two samples were mixed into one cDNA library prior to hybridization with the array.

(1) The recommended hybridization temperature for homologous hybridization of the arrays is 68°C, but we have found that the heterologous hybridization temperature must be lower. For hibernating mammals, 68°C hybridization also produces a signal, but 55°C hybridization is better. We finally found that 44°C in Church's buffer overnight gave the best results. For heterologous crosses with non-mammalian species, lowering the temperature to 40°C produces the best hybridization signals with very low background.

(2) After hybridization, the wash step needs to be adjusted and monitored to ensure that there is no loss of hybridization signal in the heterologous hybridization system. Cleaning begins with 5 X SSC (diluted with 20 X reservoir solution), 1 % SDS, then 2 X SSC, 1 % SDS, then 1 X SSC, 0.5 % SDS, and finally 0-5 X SSC, 0-5 % SDS. After each cleaning step, the ATLAS™ Array needs to be tested for hybridization signal strength using a Geiger counter. Hybridization signal strength. If the signal drops to 500-1000 cpm, the cleaning should be stopped immediately and the ATLAS™ Arrays should be exposed to X-rays or radiographic plates. Once the negative has been developed or the plate has been scanned and read, two negatives or two images (control and experimental) can be stacked and first visually screened for differentially expressed genes. For more quantitative results, convert each image from the plate or x-ray into a .tiff file that the software can analyze.

(3) ATLAS™ arrays can be reused at least 3 times. The arrays are eluted by boiling in 10% SD S for 10 min, then removing the SDS with 2 X S S C. Wrap the arrays in cellophane and store at -20°C until reuse.

(1) Prepare the hybridization solution. For each hybridization reaction, take 100 μL of DIG Easy Hyb solution and add 5 μL of fermentation tRNA (10 mg/mL) (Invitrogen) and 5 μL of calf thymus DNA (10 mg/mL) (Sgma) to minimize nonspecific binding. The mixture was heated at 65°C for 2 min and cooled to room temperature.

(2) Add 80 μL of prepared hybridization solution to the Cy3-Cy5 labeled cDNA samples. Heat the mixture at 65°C for 2 min and cool to room temperature.

(3) When using the 19 K Human Microarray, be very careful when adding the prepared probes to the slides because the gene sites are on two slides. Place one slide on top of the other, with both slides facing inward on the side with the array dot. Carefully add the hybridization solution containing the probe mixture slowly and evenly along one of the sides to prevent air bubbles.

(4) Place the remaining hybridization solution in a hybridization cassette (a sealable slide cassette placed horizontally in a 37°C thermostat) to keep the hybridization system humid. Incubate the slides in the hybridization cassette at 37°C overnight. Adjustment of the hybridization temperature is not necessary.

(5) After hybridization, the hybridization solution is washed off the slides with 2XSSC, and the microarray slides are placed on a slide rack for further washing with preheated (50°C) 2XSCC, 0.1 % SDS for 10 min, and preheated (50°C) 1XSSC, 0.1% SDS for 10 min. Finally, the slides are immersed in IXSSC and washed briefly with isopropanol. Finally, the slides were immersed in I X SSC, then washed briefly with isopropanol and centrifuged at 500 g to remove unbound fluorescent cDNA. The microarrays can be scanned at two wavelengths for quantification of different fluorescence. Fluorescence intensity analysis was performed after generating two image files. If there is concern that a good hybridization signal cannot be generated due to large evolutionary differences, it is recommended that the rigor be reduced when cleaning. For example, when the cDNA used in the study has only 60 % ~ 80 % homology, it is recommended that the wash temperature be lowered to 45°C , and that only the 2X SSC step be performed. Based on our experience and that of other laboratories (40), the salt concentration in the wash solution is most critical for removing bound probes from the array.

Analysis of cDNA arrays has become easier in recent years. Our analyses are done primarily with the program Scanalyze developed by Michael Eisen, which is free ( http://rana.lbl.gov/) and can be used in conjunction with visual screening of target genes on the array. Scanalyze allows the user to enter two array images at a time, typically a scanned image generated by Cy3 hybridization and a scanned image generated by Cy5. Further information can be obtained from http://rana.lbl.gov/manuals/ScanAlyzeDoc.pdf. There are many other DN A array analysis software available, see Note 7 for details.

(1) Open the .tiff files saved from the Cy3 and Cy5 scans of the 19K cDNA array in Channel 1 and Channel 2, respectively.

(2) After loading the images, click "redraw" to adjust the gain and homogenization of each image so that they have the same brightness and intensity.

(3) Scanalyze the images by framing each 19K cDNA "dot" with a circle generated by Scanalyze. Create a new grid for each new batch of imported array images. Click "New Grid" in the Grid Control panel and select 1~32 grids.

(4) Enter the number of rows and columns, the width of rows and columns, and the height of rows and columns for each grid.

(5) Since array dot printing is sometimes not done optimally, the grids may not match the array. If this is the case, use Scanalyze's directional buttons to move and stretch the grid up, down, left, and right. When the array's grids have been adjusted so that they almost overlap on each image, press "refine" and Scanalyze will resize the grids to the most appropriate size. If there is a mismatch of selected points, these points can be matched individually by using the "spot" option and the direction buttons.

(6) When the grid has been adjusted to fit the array, click the save data button. Scanalyze will calculate the output information of each point on the array, and the result will be exported in tab-paging format, which can be opened in Microsoft Excel.

(7) By far the fastest and easiest way to analyze the hybridization signals generated by Channel I : Channel 2 is to measure the ratio of the hybridization signals generated by Channel I : Channel 2 (e.g., control vs. hibernation group). This analysis gives an overview of the comparison of gene levels in the two states. Since the data is exported to Microsoft Excel, it is possible to sort the ratios from high to low (or low to high) (click on "Data" and select "Sort"). The points in the array with the most pronounced up- or down-regulation are displayed, and their corresponding genes are identified for subsequent analysis (see Note 8).

(1) For each screened target gene, download homologous gene sequences from NCBI (www.ncbi.nlm.nih.gov) in other animals.

(2) Select "nucleotide" from the drop-down menu and enter the name or abbreviation of the target gene.

(3) Once the sequence of a gene is obtained, it is easy to search for homologous genes in other species using the Blast database (www.ncbi.nlmnih.gov/BLAST/ ). Download the homologous sequences of the target gene in multiple species. For example, when studying squirrel genes, sequences from other rodents and/or lactating animals (e.g., mice, rats, and humans) can be selected. For example, for hibernating animal genes, we generally identify homologous regions in human (Homo mice (MmswzmscmZ ms) and brown mice (Brothers). For animals that are more distant in evolutionary distance, it is more efficient to select more diverse genes and/or genes from species that are phylogenetically closer to the target species. For example, when analyzing a gene from a sea turtle, it is more appropriate to select sequences from frogs, chickens, and rats for initial analysis. Generally, three homologous sequences are sufficient, but for a more in-depth study of a gene, multiple sequences may be selected for analysis.

One of the early concerns about DN A array analysis was the lack of public domains that could harbor the vast amount of information generated by research (48-50). The solution was to create special public databases through which researchers could have access to large amounts of microarray data for free use, thus facilitating the rapid development of some seemingly unrelated fields. For example, a researcher studying a particular gene could query a wide variety of microarray data to determine the spatial and temporal expression of that gene, and thus formulate hypotheses about the regulation of genes associated with other genes. This is precisely the purpose of the Gene Expression Database (GEO) (http://www.ncbi.nlm.nih.gov/geo/ ) introduced by the NIH (51, 52). There are also other microarray databases, such as the Standford University Microarray Database [StandfordUniversity Microarray Database (http:/ 7 genome-www5. Stanford,edu/)], which lists public data, references, and species from which the data were derived; the European Institute for Bioinformatics (EIB); and the European Institute of Biomics (EIB). EuropeanBioinformaticsInstitute's ArrayExpress database ( http://www.ebi.ac.uk/arrayexpress/) (53-56), which is functionally similar to the NIH GEO database and includes data on more than 12,000 hybrids covering at least 35 species. The GEO database is similar in function to the NIH GEO database and includes data on more than 12,000 hybridizations covering at least 35 species. GEO is by far the largest and most comprehensive open database, providing scientists with free access to high-throughput data on mRNA expression, genomic DNA analysis, serial analysis of gene expression (SAGE), mass spectrometry, and proteomics. While these databases are very useful, especially for model organism researchers, they have only recently begun to be used by those working in comparative studies.
It is clear that through cross-species heterologous microarray analysis, comparative researchers are able to greatly improve their research output. The following two situations preclude the use of cross-species heterologous array screening methods: (1 ) too little homology between the array and the target species genes; and (2) newly discovered genes that are specific to a particular species, and thus have no counterpart homologs on commercialized arrays. However, more and more new species arrays are being produced all the time, so to some extent both of these problems can be overcome when there are available arrays for species that are phylogenetically close to the target species. In the field of hibernation research, for example, the Matt A n d rew s Laboratory (5 7 ) recently prepared a DN A array using more than 4,000 cDNAs from the Mexican yellow rat (S. irzWecemGweatos) cDNA library, and used this array to perform an analysis of the transcriptome in the heart during hibernation. This array was used to analyze the transcriptome in the heart during hibernation. Because only 4000 genes were spotted on the array and a large portion of the genome was not listed, the results obtained from heterologous hybridization are more generalizable at this time, as we
For example, we hybridized ground squirrel cDNA to a commercial human array of 19,OOO genes, and the degree of hybridization was 8 5 % to 90 %. However, species-specific arrays have the opportunity to identify new genes that are only present in hibernating animals (i.e., genes that are not present in the human genome), and thus species-specific arrays have an irreplaceable role in the genetic analysis of hibernating phenotypes.
Since array screening is inevitably followed by rigorous follow-up analyses regardless of the array platform used, this is an advantage for comparative biologists using heterologous cDNA array hybridization, especially if the target species has a high degree of homology to the model species. As biologists around the world are sequencing the genomes of nontraditional model species, including the Mexican weasel, which was designated for whole-genome sequencing by the Human Genome Project, the field of heterologous microarray analysis is bound to grow considerably in the coming years. The annotation and analysis of genes and gene structures across multiple species will open up the possibility of precise gene identification and homology analysis, and further confirm the important value of heterologous microarrays for future cross-species work by comparative biologists.

Caveat

(1) An important part of any array study is the identification of experimental routes and appropriate controls. In all scientific endeavors, the selection of appropriate control conditions is a key factor in properly interpreting changes in gene expression under treatment conditions. This seems particularly important in array screening studies, which analyze mRNAs, which have a very short half-life in the cell, so it is desirable for array studies to select controls and experimental samples that are as similar as possible in time and pre-treatment state, as exemplified by one of the existing debates in the field of hibernation research. We wanted to understand the regulatory mechanisms of hibernation and what genes need to be upregulated to help animals enter hibernation and/or stabilize their metabolism to survive the long hibernation period ^ so we chose control and experimental animals that were as similar as possible: in this experiment, the controls were animals with a body temperature of 37°C that were in a room at 5°C but had not yet entered hibernation, and the experimental animals were animals that were in the same room that had already entered hibernation: the controls were animals that were in the same room that had already entered hibernation. In this experiment, the control group are animals with a body temperature of 37°C, which are in a room of 5°C but not yet in hibernation, while the experimental group are animals in the same room which are already in hibernation and whose body temperature is close to room temperature. In this way, we can obtain the difference in gene expression between the active and hibernating states. In contrast, some other groups have advocated comparing animals that are active in the summer with those that enter the hibernation phase in the winter (58). This would reflect seasonal differences in organ mRNAs but is inappropriate for studies of hibernation regulation because there are too many differences between summer and winter animals, including environmental conditions (e.g., photoperiods and thermoperiods), physiological states (e.g., active or inactive feeders, active or inactive on the ground in summer versus sleeping in burrows in winter hibernating), and reproductive states. reproductive status. Such differences make it impossible to "cut out" hibernation-specific gene expression changes and study them separately. Thus summer-active animals are arguably an extremely poor biological control, and even more arguably the wrong time of year, and a devastating factor in the overall study of hibernation. Indeed, a genetic screen applied to our experimental system (constant temperature versus hibernating animals) showed that the expression of a large number of genes was specifically induced when the animals entered the hibernation period; these genes appeared to perform essential biological functions in the hibernating state. We also found a wide range of organ-specific activation of stress-induced signaling pathways in the hibernating group compared to the thermostable control group, including mitogen-activated proteins (MAPs) and mitogen-activated proteins (MAPs).This included mitogen-activated protein kinase (17, 59), suggesting that basic metabolic activity is maintained in vivo during hibernation. These findings are actually contrary to the so-called 'conventional experience' of some previous hibernation studies, which suggest that most biological processes are attenuated or simply cease during hibernation.(2) Nota

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