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The introduction of large-scale gene expression profiling studies has greatly increased the need to rapidly obtain high-quality cellular expression patterns of genes found to exhibit differential expression. The use of large-scale nonradioactive RNA in situ hybridization makes this possible, and greatly increases the general usefulness of this data. Here, we describe protocols for parallel analysis of up to 50 different gene-specific probes in mouse retinal sections.
RNA in situ hybridization (1, 2) can be used to characterize the cellular expression pattern of any RNA species. By designing anti-sense probes that can undergo complementary base pairing with a target sequence of interest, one can readily design a probe that can strongly and selectively bind to virtually any target RNA, whether or not it codes for protein. The high stability of RNA–RNA hybrids means that hybridization conditions can be made especially stringent, thus resulting in both low background signal and high sensitivity. Complementary RNA (cRNA) probes are typically generated from linear DNA templates, using recombinant viral RNA polymerases to directly incorporate modified bases that contain a label of choice to allow detection of probe–target hybrids. Radioactive nucleotide triphosphates can be used to allow for direct detection of bound probe. Alternatively, chemically modified bases can be used, allowing indirect probe detection using immunodetection. Critical improvements came with the use of bases conjugated to the highly antigenic small molecule digoxigenin for probe labeling, which allowed the use of highly specific alkaline phosphatase-conjugated antibodies for immunohistochemistry (2, 3). These modifications improved the specificity and sensitivity of the protocol to the point where it could be used to analyze many different probes in parallel. One of the most spectacular uses of this approach has been the effort of the Allen Brain Atlas consortium to map the expression of all annotated mouse genes in the adult brain (4).
In the retina, large-scale in situ hybridization has been instrumental in analyzing the results of SAGE (5, 6) and microarray (7, 8) data obtained by profiling different developmental stages, or mutant animals exhibiting developmental defects or retinal degeneration. While global expression profiling of this sort generates vast amounts of data, it is very hard to interpret meaningfully unless one also knows the cellular expression pattern of any differentially expressed genes. Even in cases where isolated cell subtypes or even individual cells are profiled (9, 10), it is important to use a different experimental approach to confirm the validity of any results obtained. The regular structure of the retina enables the major cell types that express a given gene to be identified on the basis of their laminar position, making this data particularly useful. Since up to 50 different cRNA probes can be run in parallel by a single investigator, with results obtained within 3–5 days, it is now feasible to rapidly sort through all of the most potentially interesting hits obtained in such experiments.
Carry out all procedures at room temperature unless otherwise specified.
MilliQ water is adequate for this and all subsequent steps.
1DEPC is highly toxic. Use caution when preparing solutions, and do not breathe vapor. Autoclaving will fully inactivate 0.1% DEPC, and the faint smell detectable after autoclaving reflects residual ethanol contamination.
2DEPC reacts with amine, hydroxyl, and thiol groups of proteins, thus inactivating RNAse, as well as any other protein with which it comes into contact. As a result, it is highly toxic and should be handled with great care.
3The filter unit used to prepare B3 can be wrapped with parafilm and reused.
4The viscosity of the Gelvatol requires optimization for each batch prepared. When making the solution, add PVA in step 4 until the solution is clear and is slightly less viscous than molasses. Then refrigerate the beaker of Gelvatol overnight at 4°C (after step 5 and before step 6), and check it the next morning to be sure that the viscosity is that of molasses. If it is, continue on to step 6. If it is too viscous, add a little more glycerol to lower the viscosity and then go on to step 6. If it is not viscous enough, add more PVA with heat and refrigerate for a few more hours, again checking the viscosity before going on to step 6. Continue until viscosity is optimal. Gelvatol can be stored at 4°C indefinitely, or at room temperature for 1 month.
5DNA templates used for cRNA probe synthesis should be between 300 and 2,000 bp in length, with 700–1,000 bp being optimal. Probe sequences should lack any repeat sequences longer than 40 bp, and should not show more than 90% identity over any continuous stretch of 150 bp or more, or cross-reactivity will result. Probe templates must be cloned into vectors (such as pBluescript) in which the insert is flanked by T7, T3, or Sp6 RNA polymerase promoters, so as to allow generation of labeled cRNAs. Many 3′ directed ESTs, such as those from the BMAP project or other large-scale cDNA sequencing efforts, work very well as ISH probes, and can be easily ordered from companies such as Thermo-Fisher. Alternatively, PCR amplification followed by cloning into an appropriate vector (such as the TOPO-TA vector) can be used if repeat-free ESTs are not already available as probe templates. Only cloned sequences should be used as templates for probe generation. While PCR can be used to simultaneously amplify template sequences from complex target preparations (e.g., reverse-transcribed cDNA), this introduces a strong possibility of contamination. Once a clone containing the probe sequence has been obtained, a linearized template must be generated in order to conduct run-off transcription for cRNA synthesis. All experiments should include both a positive and a negative control sample. Positive control probes should robustly recognize target mRNAs in retina, but not be so abundant that contamination of other slides is a real risk. Good negative control probes should target transcripts that are not detectably expressed in retina (such as albumin, cardiac albumin, GFP, etc.). Use of sense control probes is not usually recommended, as a sizeable minority of mammalian genes have associated antisense transcripts (11).
6When using PCR primers that target an RNA polymerase promoter sequence (i.e., T3, T7, or Sp6), it is important to include 2–3 bases of 5′ overhang outside the primer sequence. This substantially improves the efficiency of cRNA synthesis.
7Fixed tissue generally produces weaker signal intensity than fresh-frozen, though use produces superior morphology and better overall signal–noise ratio. Fresh-frozen tissue is often easier to section consistently than fixed tissue, and is particularly useful for analyzing embryonic samples.
8It is advantageous to fill as much as the slide as possible with sectioned samples (although make sure that O.C.T. from other sections does not overlap your tissue). The more sections present on a slide, the greater the likelihood of obtaining high-quality data in a given experiment. It is also the best way to directly compare results from different samples, since placing them on the same slide effectively eliminates slide-to-slide variation in signal intensity and probe spreading.
9RNAses are very resistant to inactivation, so pretreatment is designed to simply denature and remove any proteins that might come into contact with the slides. In this step, this is accomplished by treatment with detergent and a strong base.
10Avoiding RNAse contamination during the first day of the procedure is critical. RNAses can come from human and animal skin and hair, or very often as contamination from other experimental procedures, particularly plasmid DNA preparation. The danger is greater to the probe than to the sample, as target RNA sequences in fixed tissue are much less accessible to RNAses than are cRNAs in solution. Do everything possible to avoid these sources of contamination. Wear a clean lab coat to cover arms, tie back long hair, and avoid working in lab areas where animals are sacrificed. Use aerosol tips for all solutions that require pipetting. Put down fresh bench paper before beginning the procedure, and change gloves whenever the possibility of contamination arises. We have often used a fume hood to conduct all the steps up until the prehybridization step, which both protects your sample from airborne debris and removes any PFA vapors from the work area.
11Dunk sections several times during each wash to ensure good mixing.
12This refixation is important to maintain tissue integrity following the protease K treatment.
13Acetic anhydride modifies positively charged amine groups in proteins and lipids, greatly reducing nonspecific binding by the negatively charged cRNA probe. However, acetic anhydride hydrolyzes rapidly in aqueous solution, and it is extremely important that the solution be applied immediately to the slides once mixed.
14It is important that slides rest above the surface of the box so as to avoid capillary transfer of solution away from the sections.
15Do not allow the tissue to dry out at this or any other successive stage. This will result in a loss of signal in the affected area. Chill prehybridization solution on ice prior to applying to reduce surface tension and ensure an even spread. Solution used for prehybridization can be reused 3–4 times. Pour into 50 ml conical tubes prior to adding probe and store at −80°C (reused solution should not be mixed with probe and used for hybridization).
16Probe must be well mixed to ensure even signal intensity across the slide.
17It is critically important to change gloves between probes. Failure to do so will result in cross-contamination.
1870–72°C is an optimal hybridization temperature for perfect match probes that are longer than 300 bases. 65°C is better for shorter probes or an imperfect match (i.e., rat probe used on mouse tissue).
19Do not use any force or pressure to remove the coverslip. Doing so will result in damage to the tissue.
20In this and every subsequent step, preheat solutions in a water bath prior to washing.
21The RNAse treatment degrades the single-stranded cRNA probe while leaving double-stranded RNA–RNA hybrids intact. This results in a considerable reduction in background signal. However, it requires great caution, as the RNAse used in this step can contaminate materials used prior to the hybridization step. Every effort should be made to prevent this. Make sure that slide boxes and racks used for RNAse treatment are treated with 0.3 M NaOH and RNAseZap after use. Change gloves after handling slides during this step.
22To ensure even spread of solution across the slide and to reduce the amount of serum used, chill B2 solution on ice prior to application.
23For abundant RNA a 1-h incubation is sufficient, but overnight incubation greatly enhances the signal and reduces color reaction time.
24Optimizing the ratio of target to background signal is crucial for obtaining high-quality data using this procedure. To this end, it is essential to vigilantly track the progress of the color reaction so as to assess when it should be terminated. The most efficient way to do this is to directly view the slides in their chamber against a white background using a dissecting microscope such as a Leica Stemi 2000-C or an Olympus BH-2 at roughly 10× magnification. Stop the reaction before any obvious background is visible, but slightly past the point at which the color exposure appears optimal to the eye, as the cellular signal intensity will look weaker at the 200× magnification usually used for photographing retinal sections.
25Gelvatol automatically seals the slide once it dries, which reduces labor substantially when running large number of slides. One can also use Aquamount (Fisher, 14-390-5) or an equivalent water-based solution to mount the slides, but they must then be sealed with clear nail polish to prevent drying during long-term storage.