Whitfield, C. W., Cziko, A.-M., and Robinson, G. E. (2003). Gene expression profiles in the

brain predict behavior in individual honey bees. Science 302, 296–299.

Wolfinger, R. D., Gibson, G., Wolfinger, E. D., Bennett, L., Hamadeh, H., Bushel, P., Afshari,

C., and Paules, R. S. (2001). Assessing gene significance from cDNA microarray

expression data via mixed models. J. Comput. Biol. 8, 625–637.

Yang, Y. H., and Speed, T. (2002). Design issues for cDNA microarray experiments. Nat. Rev.

3, 579–588.

Zimmer, D. P., Soupene, E., Lee, H. L., Wendisch, V. F., Khodursky, A. B., Peter, B. J.,

Bender, R. A., and Kustu, S. (2000). Nitrogen regulatory protein C-controlled genes of

Escherichia coli: Scavenging as a defense against nitrogen limitation. Proc. Natl. Acad. Sci.

USA 97, 14674–14679.

[32] gene expression techniques in plants 617

[32] Methods for Studying the Evolutionof Plant Reproductive Structures:

Comparative Gene Expression Techniques

By Elena M. Kramer

Abstract

A major component of evolutionary developmental (evo-devo) genet-ics is the analysis of gene expression patterns in nonmodel species. Thiscomparative approach can take many forms, including reverse-transcrip-tase polymerase chain reaction, Northern blot hybridization, and in situhybridization. The choice of technique depends on several issues such asthe availability of fresh tissue, as well as the expected expression level andpattern of the candidate gene in question. Although the protocols for theseprocedures are fairly standard, optimization is often required because ofthe specific characteristics of the species under analysis. This chapter de-scribes several methods commonly used to determine gene expressionpatterns in angiosperms, particularly in floral tissues. Suggestions foradapting basic protocols for diverse taxa and troubleshooting are alsoextensively discussed.

General Considerations for Working with RNA

RNA is, by nature, a less stable molecule than DNA and there aremany sources of RNase in the environment, which can lead to its rapiddegradation. These facts often lead to trepidation on the part of research-ers who are not experienced with RNA work. However, such concerns arelargely unnecessary because simple precautions can effectively prevent

Copyright 2005, Elsevier Inc.All rights reserved.

METHODS IN ENZYMOLOGY, VOL. 395 0076-6879/05 $35.00

618 functional analyses [32]

RNA degradation. First, researchers must always wear sterile gloves whenworking with RNA. Gloves should be changed if the skin or items generallyhandled without gloves, such as doorknobs, are touched. Many labs go sofar as to have specific RNA-use benches or even rooms, but this is not anabsolute requirement as long as the bench space is kept clean. Second, allplasticware (tips, pipets, tubes) should be purchased RNase-free and keptthat way by maintaining them as separate lab stocks that are handled onlywith gloves. For instance, RNase-free supplies can be stored in a separatecabinet that is kept locked to prevent unintentional contamination. Third,glass or metalware should be made RNase-free either by baking at 235� formore than 2 h (careful baking bottle caps, most will melt!) or by treatingwith 0.1 M NaOH overnight followed by thorough rinsing with sterilewater. Before baking, wrap bottle mouths and metal items, such as slideracks or stir bars, in aluminum foil to help keep them RNase-free afterbaking. Fourth, all solutions should be made with water, chemicals, stirbars, cylinders, bottles, and others, which are all RNase-free. RNase-freewater can be prepared using diethylpyrocarbonate (DEPC), which is highlytoxic and should be used only under a fume hood. DEPC is typicallydiluted in double-deionized water (ddH2O) to produce a 0.1% solution.Because DEPC is unstable once the bottle seal is broken, it is best toprepare large batches of DEPC water at one time in order to use the entirevolume. Take clean bottles of various sizes (100 ml to 2 L), fill with fixedamounts of ddH2O, and place under a fume hood. Aliquot the appropri-ate amounts of DEPC into each bottle, tightly secure the bottle caps, andshake. Let the bottles sit in the fume hood for 2–24 h. DEPC has only a30 min half-life in ddH2O, but autoclaving for 15–30 min will ensure thatall DEPC is inactivated. It is important to note that DEPC can be addedto many solutions directly, but it is not compatible with Tris or MOPSbuffers. Tris and MOPS solutions can be prepared using DEPC water, andRNase-free bottles and chemicals so treatment is not necessary after solu-tion preparation. Also, remember that the bottles that are used to makeDEPC water can also be treated as RNase-free themselves after they areemptied. See http://www.ambion.com/techlib/basics/rnasecontrol/ for moreinformation on DEPC water and RNase control methods.

Methods Based on RNA Extraction

Collecting and Storing Tissue

Given the instability of RNA, the collection and storage of tissue be-comes a much greater concern than when the goal is DNA extraction. Thereare two primary methods for storing tissue for future RNA extraction:

[32] gene expression techniques in plants 619

freezing at �80� or infiltration with a special RNA preservative such asRNAlater (Ambion). In either case, it is critical to quickly collect thematerial and complete the selected treatment. Some tissues may be moresensitive to RNA degradation than others, but no more than 15 min atambient temperature or 1 h on ice is ideal. For best results, tissue should betreated immediately. In cases in which dissection is required, material canbe kept on ice during the dissection and the separated tissues treated assoon as the desired amount is obtained (but observing the time framementioned earlier).

For freezing, it is best to collect tissue in plastic screw-top or snap-toptubes. Plastic or paper bags perform poorly during long-term storage at�80�. Make sure to mark all samples with ink or labels that are compatiblewith cold storage. Once material is frozen, it is critical that it be maintainedat temperatures less than �50�; RNA will not tolerate freeze–thaw cyclesof more than 30�. Material can be stored at �80� for very long periods,however, as long as the temperature is controlled.

As a general rule, tissue that has been fixed in formaldehyde or similarchemical fixatives is not suitable for RNA extraction. Likewise, silica-preserved tissues will not yield usable RNA. These issues must be takeninto consideration when planning any experiment that will use RNA and,unfortunately, often mean that remote field collections are not compatiblewith RNA-based techniques.

Methods of RNA Extraction

Many expression analysis techniques, such as Northern blot hybri-dization and reverse-transcriptase polymerase chain reaction (RT-PCR),start with the extraction of total RNA. Although numerous plants areperfectly amenable to this process, it is not uncommon for the presenceof polyphenols, polysaccharides, mucilage, and other compounds to in-hibit RNA extraction from plant tissues. A number of specialized protocolshave been developed to deal with particularly recalcitrant species ortissue types (Chang et al., 1993; Suzuki et al., 2003; Wang et al., 2000).As a general rule of thumb, younger tissues, such as flower buds or im-mature leaves, are easier to work with. Many kits and products forRNA extraction are available (see http://www.ambion.com, http://www.qiagen.com, and http://www.invitrogen.com, among others). We prefer touse Concert Plant RNA Reagent by Invitrogen (Carlsbad, CA) because ofthe simple scalability of the protocol for various amounts of tissue (100 mgto 5 g). This protocol is not reproduced here because we follow the manu-facturer’s instructions quite closely, but a number of general considerationsare noteworthy.

620 functional analyses [32]

. It is unnecessary to treat items such as mortars and pestles orspatulas to make them RNase-free. Although they should be clean, there ismuch more RNase in the tissue than there is on these tools.

. The best way to prevent degradation of RNA during the grindingprocess is to keep all utensils very cold. This will also aid in grind-ing. Prechill mortars and pestles with liquid nitrogen for 5 min beforegrinding the tissues in liquid nitrogen. Grind small amounts of tissue inmicrotubes using disposable micropestles (e.g., VWR KT49521-1590)prechilled in liquid nitrogen.

. Best results will be obtained by grinding the frozen tissue to adustlike consistency. This should be rapidly transferred to a large tubecontaining the appropriate amount of extraction buffer using a prechilledspatula. For small amounts of tissue, further maceration of material in theextraction buffer can greatly increase yield.

. Once an RNA pellet has been obtained, rinse it carefully in 70%ethanol made with diethylpyrocarbonate (DEPC) water. The dry pelletshould become translucent and glassy.

. Resuspension of the RNA is aided by the use of prewarmed (50–60�)RNase-free water or 2 mM of ethylenediaminetetraacetic acid (EDTA)pH 8.0. Resuspend the pellet in as small a volume as possible to facilitatedownstream protocols. Inability to resuspend the pellet may result fromoverdrying (never dry RNA in a SpeedVac) but is also often indicative ofthe presence of secondary compounds or starch. This may indicate thatalternative preparation protocols will be necessary.

. RNA should be stored at �80�.

Northern Blot Hybridization

Northern blots can be useful to examine the expression of genes invarious tissue types or over a range of developmental stages. They canbe prepared using either total RNA or poly(A) RNA. Several standardprotocols for Northern blot preparation are available, including that ofSambrook and Russel (2001a). General tips for performing Northern blothybridization are as follows:

. Prepare the electrophoresis apparatus, gel casting tray, and gel combby treatment with 0.1 M NaOH for 12 h, followed by thorough rinsingin water.

. The RNA must be suspended at a reasonably high concentration toallow loading of a large amount of RNA (5–10 �g) in 1–5 �l of sample.Care should be taken to equalize the loading amount of each sample sodirect comparisons can be made across different lanes.

[32] gene expression techniques in plants 621

. Once the RNA transfer to the membrane is complete, the transferefficiency can be checked by illuminating the blot with a handheld ultraviolet(UV) light source. The nucleic acids must be fixed to the membrane surface,which can be done using commercially available UV cross-linkers or bybaking in a vacuum oven for at least 1 hr at 80�. Using both methods increasesthe stability of the blot and facilitates repeated probing and stripping.

. I recommend a hybridization solution for Northerns composedof 50% formamide, 3� SSC (3.0 M NaCl, 0.3 M sodium acetate), 0.5%sodium dodecyl sulfate (SDS), 0.1 mg/ml of herring sperm DNA, 5XDenhardt’s solution (1% Ficoll, 1% polyvinylpyrrolidone, 1% bovineserum albumin [BSA], filter sterilized), and 25 mM of ethylenediami-netetraacetic acid (EDTA) pH 8. This increases the stringency of thehybridization.

Blots prepared in this manner can be stripped and hybridized withdifferent probes up to six times. Relative lane loading can also be assessedby hybridizing the blot with a control probe, such as ubiquitin or actin, inthe final hybridization. When combined with phospho imaging, this canallow more accurate quantification of relative expression levels.

PCR-Based Methods

To an increasing degree, PCR-based approaches are replacing the use ofNorthern blots to assess gene expression. These techniques include ‘‘semi-quantitative’’ RT-PCR and quantitative or real-time RT-PCR (RT-qPCR).Before PCR can be conducted, first-strand cDNA must be synthesized fromthe RNA. Although total RNA can be used as the template for this reaction,I have found that using poly(A) RNA yields the best results. A caveat to thisstatement, however, is that poly(A) RNA constitutes only about 10% of atotal RNA extraction, and typical yields are in the range of 1–5%. This meansthat extraction of poly(A) from total RNA is worth pursuing only whenstarting with a fairly large amount of total RNA (at least 50–100 �g). Manypoly(A) extraction kits are available, including some that allow direct prepa-ration of poly(A) from tissue (see Ambion, Qiagen, Invitrogen, Clontech). Iprefer to use magnet-based methods (Dynal, Ambion, Novagen), whichallow the elution of poly(A) RNA in very small volumes.

Before cDNA synthesis, RNA should be treated with RNase-free DN-Ase, particularly when working with total RNA. The stop solutions typi-cally used with DNAse, however, contain concentrations of EDTA thatmay inhibit further enzymatic reactions. This means that treated RNAmust be column cleaned using a product such as RNAqueous (Ambion,Austin, TX) or RNeasy (Qiagen, Valencia, CA) and eluted in DEPC water.

622 functional analyses [32]

RNA can be easily quantified with UV spectrophotometry; high-qualityRNA should give an A260/A230 reading of more than 1.8. Electrophoresis ina typical TBE or TAE gel is also useful to assess RNA quality. Keeping asmall electrophoresis rig, gel casting tray, and gel comb RNase-free is usefulif this is going to be performed frequently. Good quality total RNA willappear as a long, bright smear punctuated by two to four distinct bands ofmoderate molecular weight, which represent the rRNAs and sometimesrbcL transcripts if the tissue is photosynthetic. Poly(A) RNA does not looklike much on a gel, just a bright smear in the range of 400–2000 bp. A faintsmear with a bright spot at low molecular weight (<100 bp) indicates thatthe RNA is degraded.

Preparation of first-strand cDNA can be done in a separate reaction orat the same time as the PCR (one-step RT-PCR). Again, many manufac-turers supply products for both types of procedure (Ambion, Invitrogen,Stratagene, Clontech, Promega, etc.). Because my laboratory is typicallyinterested in the expression of multiple genes, my colleagues and I prefer toprepare cDNA in a separate reaction using an anchored poly-T primer [seeKramer et al. (1998)]. This type of cDNA is stable at �20� for severalmonths and can be used with any combination of degenerate or specificPCR primers. Following cDNA synthesis, the reaction can be treated withRNase to remove all remaining RNA, and the cDNA can be columnpurified using any available PCR cleanup kit. The best way to assess thesuccess of the cDNA reaction is to use it as a template in a PCR withprimers for control loci such as ubiquitin or actin.

Semiquantitative RT-PCR simply involves performing PCR on cDNAusing primers that are designed to be specific to the gene of interest. It isadvisable to design the primers so that they span at least one intron. Thisallows the amplification of contaminating genomic DNA to be simplydetected because the product derived from genomic DNA will be largerthan that derived from cDNA. In addition to the typical concerns fordesigning PCR primers (annealing temperature, GC content, etc.), primerspecificity should be carefully considered so that only the gene of interest isamplified, particularly when working in large gene families. Every effortshould be made to use equivalent amounts of cDNA in each reaction, andit is important to run positive control reactions using primers for a genethat is expected to be expressed fairly uniformly, such as actin.

Several caveats are important to consider when performing semiquan-titative RT-PCR and interpreting the results. The highly sensitive natureof PCR means that even samples that have vanishingly small amounts oftarget can yield bands if subjected to enough amplification cycles. For thisreason, it is advisable to use cycle numbers in the 20–25 range to increase

[32] gene expression techniques in plants 623

confidence that the detection of cDNA is significant. Of course, some lociare expressed at such low levels that additional cycles are necessary. In thiscase, it is preferable to show the results for several experiments using bothlow and high cycle numbers. Real-time or quantitative RT-PCR is rapidlybecoming a standard method for more accurate analysis of relative geneexpression levels. These methods require specialized equipment, includinglight-detecting PCR machines. The technical considerations and data ana-lyses involved in this procedure are quite complex and beyond the scope ofthis chapter. Several excellent web sites on this topic exist, including http://www.wzw.tum.de/gene-quantification/. For both semiquantitative and RT-qPCR, however, several limitations remain. Early stages of developmentare often difficult to analyze, because of the inability to dissect separateorgans for RNA extraction. In addition, important aspects of gene expres-sion, such as spatial patterns of RNA distribution within an organ, are notobservable. For these reasons, the best tool for assessing gene expressionpatterns remains in situ hybridization.

In Situ Hybridization

In situ hybridization is, as already mentioned, the best available methodfor obtaining information on the specific spatial and temporal patterns ofgene expression in developing tissues and organs. At the same time, themethod is complex and requires considerable preparation and expertise.Commonly used protocols involve hybridization of radioactive or nonra-dioactive RNA probes to sectioned tissue. Radioactive probes are thoughtto be more sensitive but have many drawbacks, including poor localiza-tion of the signal, instability of the probe, and the requirement to processslides in complete darkness. Several protocols for labeling and hybridi-zation of radioactive probes are available, including that of Weigel andGlazebrook (2002). Nonradioactive in situ hybridization is generally pref-erable and is described here. Many variations on the protocol exist, but allare derived from Jackson (1991). The following protocol has been success-fully used with magnoliid dicot, monocot, and eudicot floral tissue. Theprocedure is described in six parts: fixation and embedding of tissue,preparation of RNA probes, preparing for in situ hybridization, sectioningand prehybridization, posthybridization, and imaging.

Fixation and Embedding of Tissue

As with tissue collected for RNA extraction, tissue intended for ultimateuse in in situ hybridization must be carefully processed.

624 functional analyses [32]

1. Dissect or collect tissue and immediately submerge in ice cold,freshly prepared FAA (50% ethanol, 10% formalin, 5% acetic acid). It iscritical to use freshly prepared FAA and preferable to use formalin that isnot more than a year old.

2. Place the tube containing the FAA and tissue into a beaker filledwith ice, and place this into a desiccator. Vacuum infiltrate the tissue for1.0–1.5 h. Hold the vacuum for 15 min and then release slowly. Repeat.Tissue may sink, and many protocols suggest that this is a necessity.However, it is common for plant materials that are very pubescent (hairy)to remain buoyant in FAA. It is more important not to overfix your tissuethan to wait for it to sink.

3. Following vacuum infiltration, refill the tube with fresh FAA asnecessary and place it on an oscillating shaker at 4�. Protocols vary as tohow long tissue should be incubated in fixative after vacuum infiltration.Very small herbaceous tissue can be immediately removed from the fixa-tive following vacuum infiltration and dehydrated. Larger, dense tissuetypically requires 4–12 h of incubation in FAA for proper fixation. Neverfix tissue for more than 16 h total.

Note: Many people are not aware that tissue can be overfixed. This is aserious problem that must be avoided for optimal in situ hybridizationresults. Tissue that has been in fix for more than 24 h is unlikely to beuseful for in situ hybridization.

4. Dehydrate the samples through the following ethanol series at 4� withagitation for 30-90 min each: 50% ethanol, 70% ethanol, 85% ethanol, 90%ethanol, 100% ethanol. The duration of the incubation depends on the natureof the tissue. Similar to the case with fixation, small herbaceous tissue re-quires shorter periods than denser tissues. Tissue can be stored overnight orfor longer periods at 4� during the 70% ethanol stage. After the 100% ethanolstep, exchange the solution for fresh 100% ethanol and leave overnight at 4�.

5. Exchange the solution for fresh 100% ethanol and incubate at roomtemperature (RT) for 1 h. Follow this by subsequent RT incubations in50% ethanol/50% Citrisolv (Fisher Scientific 22-143-975) for 2 h, then100% Citrisolv for 2 h.

6. Transfer tissue to a small glass beaker or scintillation vial and addjust enough fresh Citrisolv to cover the tissue. Fill the beaker with Para-plast Plus chips and incubate overnight at 55–60�. For the next 2 days,exchange the Paraplast with fresh molten Paraplast two to three times eachday. Material must be maintained at 55–60�. With dense tissue or organsthat tend to retain air bubbles (such as spurs), vacuum infiltration of themolten Paraplast may be necessary. This can be accomplished using avacuum oven set to 60�, applying moderate vacuum for 30–60 min.

[32] gene expression techniques in plants 625

7. Embed the tissue by filling molds (such as Peel-A-Way or Tissue-Tek)with molten Paraplast and placing individual samples in each. This can bedone on a hot plate to allow time to properly orient the tissue. Allow themolds to cool in a RT waterbath.

8. Embedded tissue can be stored for long periods at 4�.

Preparation of RNA Probes

For nonradioactive probes, antisense and sense RNA is typically la-beled using digoxigenin (DIG), which is then detected using an anti-DIGantibody conjugated to alkaline phosphatase. All DIG-labeling suppliesmentioned below are available from Roche Applied Science (http://www.roche-applied-science.com). All solutions are prepared with DEPC waterand RNase-free chemicals or treated with DEPC and autoclaved afterpreparation.

1. Chose a region of your gene of interest to serve as the probetemplate. Generally, a 200-500 bp fragment is optimal. Although in situhybridization is typically quite stringent in its specificity, it is preferable touse regions that do not show high conservation across members of a genefamily, such as DNA-binding domains. However, using solely 30 UTRsequence as template is not advisable because these regions often formsecondary structures that can interfere with probe hybridization.

2. Generate a linearized version of the template either by restrictiondigestion or by PCR. If using PCR, the RNA polymerase binding site canbe incorporated into the fragment in one of the oligonucleotide primers.PCR products should be cleaned and concentrated using a spin column. Inthe case of plasmid linearization, it is important to use a restriction enzymethat does not create a 30 overhang, which can result in nonspecific polymer-ase initiation. The chosen enzyme should cut at the end of the templateinsert opposite from the RNA polymerase binding site. Confirm completedigestion by running linearized plasmid on an agarose gel.

3. Extract the linearized DNA with an equal volume of phenol/chlo-roform, then chloroform. Use RNase-free tubes and tips following the firstextraction. Precipitate the DNA by adding 2 volumes 100% ethanol and 0.1volume of 3 M NaAC, incubating at �20� for 2 h, and centrifuging at highspeed for 10 min. Wash the pellet with ice cold 70% ethanol, dry, andresuspend in DEPC water at a concentration of about 1 �g/�l.

4. Set up a runoff transcription reaction as follows:

1 �l DNA at 1 �g/�l2 �l NTP/DIG–UTP mix2 �l 10X transcription buffer

626 functional analyses [32]

2 �l T7, T3, or SP6 RNA polymerase1 �l RNase inhibitor12 �l DEPC waterMix by gently pipetting and incubate at 37� for 2 h.

5. Set aside a 1-�l sample for later use (see step 7).6. Add 2 �l of RNase-free DNAse. Mix and incubate at 37� for 15 min.7. Take another 1-�l sample. Run this sample side by side with the

pre-DNAse sample (step 5) on a small agarose gel (use RNase-free loadingbuffer to run the samples). Bright RNA bands should be visible in bothlanes. RNA probe often runs as multiple bands. DNA template should bevisible in the pre-DNAse sample but not in the post-DNAse. If the DNA isnot eliminated, add 1 �l of DNAse and incubate another 30 min at 37�.Repeat analysis as needed.

8. Once DNAse completion has been confirmed, stop the reaction byadding 4 �l 200 mM EDTA pH 8.0. Precipitate the RNA by adding 5 �l4 M LiCl and 150 �l ethanol. Incubate at �20� for 2 h before precipitationby centrifugation for 10 min at maximum speed.

9. Wash pellet with ice cold 70% ethanol and allow to air dry.10. Hydrolize RNA probe to desired length by resuspending pellet in

50 �l of 0.1 M NaHCO3 pH 10.2. Incubate at 60� for an amount of timedetermined by the formula t ¼ (Li � Lf)/K(Li)(Lf), where t is the time inmin, K is 0.11 breaks/min, Li is the initial length of the probe in kilobases,and Lf is the final desired length in kilobases. For instance, to hydrolyze aninitial probe of 0.5 kb to 0.15 kb (the typically recommended length),t ¼ (0.5–0.15)/(0.11 � 0.5 � 0.15) ¼ 42 min. See Trouble-shooting in situhybridization section for further discussion of probe hydrolysis.

11. Stop the hydrolysis reaction by adding 5 �l 5% acetic acid, 5 �l 3 Msodium acetate, and 125 �l ethanol. Incubate at �20� for two h. Precipitateby centrifugation for 10 min at maximum speed, wash pellet in ice cold70% ethanol, dry, and resuspend in 20 �l deionized formamide.

12. Quantify probe concentration using the Roche protocol, availableat http://www.roche-applied-science.com under the title ‘‘Estimating theYield of DIG-labeled Nucleic Acids.’’ Probe can be stored at �20� forseveral months.

Obtaining a good-quality probe is very important for the success of thehybridization. Reactions that yield poor concentrations are unlikely toperform well. In my experience, T7 or T3 RNA polymerase gives betterresults than SP6. For probe templates that are particularly GC rich, it maybe necessary to prepare a special NTP/DIG–UTP mix. The DIG–RNAlabeling mix supplied by Roche has 3.5 mM DIG-11–UTP/6.5 mM dTTPand 10 mM of each remaining NTP. DIG-11–UTP and NTP solutions can

[32] gene expression techniques in plants 627

be purchased separately in order to prepare alternate concentrations, suchas 6.5 mM DIG-11–UTP/3.5 mM TTP. Higher DIG-11–UTP concen-trations will result in more DIG incorporation, but it also will reduce theefficiency of the transcription reaction. It is recommended to prepareboth sense and anti-sense probes. Anti-sense probe will be produced bytranscription from an RNA polymerase binding site at the 30 end of thetemplate fragment, whereas the sense probe is made by transcription fromthe 50 end.

Preparing for In Situ Hybridization

Preparing to perform in situ hybridization takes 3–5 days, depending onhow many solutions need to be made and your experience with the process.The following basic RNase-free stock solutions are required [refer toSambrook and Russel (2001b) for details]:

A

10� PBS5� NTE20� SSC10� PBS with 20 mg/ml glycine (store at 4�)1 M Tris pH 9.51 M pH Tris 8.01 M Tris 7.50.5 M EDTA pH 8.05 M NaCl1 M MgCl2dditional required RNase-free solutions include

10� pronase buffer: .5 M Tris 7.5, 50 mM EDTA (see the discussionon pronase, later in this chapter)

10� in situ salts: 3 M NaCl, 100 mM Tris pH 8, 100 mM NaH2PO4 pH6.8, 50 mM EDTA

Hybridization solution (800 �l): 100 �l 10� in situ salts, 400 �l deio-nized formamide, 200 �l 50% dextran sulfate (this will requireheating to dissolve), 20 �l 50� Denhardt’s solution, 10 �l tRNA(100 mg/ml in DEPC water), and 70 �l DEPC water

Note on the hybridization solution: It is best to prepare a relativelylarge volume (10–15 ml) and aliquot this into working amounts(1 ml) in RNase-free microtubes. It is absolutely critical that thissolution be RNase free, so prepare it with care.

In addition to preparing these solutions, all glassware (additional bot-tles, graduated cylinders, Coplin jars), slide racks, and stir bars must bebaked. Other plasticware, such as bottle caps, can be treated with 0.1 M

628 functional analyses [32]

NaOH as described earlier. I recommend using square polyethylene (VWR36318-045) and flat Nalgene (VWR 36212-204) boxes for incubation andwashing steps. These can be thoroughly washed twice in an automateddishwasher, once with detergent and once without. Dry upside down onpaper towels. Glass staining dishes can be similarly treated. Prepare at least8 L of ddH2O by autoclaving for 30 min in baked bottles. This ‘‘clean’’water will be used for preparation of the hydration series and all dilutedworking solutions.

Sectioning and Prehybridization

Place five to six ProbeOn Plus slides (Fisher 15-188-52) onto a slidewarmer set at about 40� and cover the unfrosted surface of each slide with apool of DEPC water. It is important to use ProbeOn Plus slides to promoteproper adhesion of the sections and allow sandwiching of slides (seebelow). Paraplast Plus embedded tissue should be sectioned at 8-�m thick-ness on a rotary microtome. Separate the ribbon of sections into strips oftwo to three sections using a clean razor blade and place sequentially ontothe slides. Small paint brushes or wooden applicator sticks can be used tomanipulate the sections. The sections must be allowed to flatten on thesurface of the DEPC water. After about 15 min, carefully remove excesswater using a Pasteur pipette around the edges of each slide. After anotherperiod of about 15–30 min, remaining water can be removed by gentlytipping the edge of slide against a stack of Kim wipes or paper towels.Repeat this process as needed in order to obtain 20–25 slides.

Sectioning can be done the day before the prehybridization, but sec-tioning on the same day generally yields better results. However, for thesections to adhere properly, it requires at least 4 h of incubation from thetime the slides are completely drained. During this period, slides shouldremain on the slide warmer with the lid closed. If you choose to section theday before, do so late in the afternoon and turn the temperature of the slidewarmer down below 30� before leaving the slides overnight.

Before starting the prehybridization, several solutions need to beprepared. First, the hydration series should be arranged using squarepolyethylene boxes, which typically hold 300 ml each. Second, prepare allthe diluted solutions, including 1.2 L of 1� PBS, 300 ml of 1� pronasebuffer, and 300 ml of 1� PBS with 0.2% glycine. These can be made bydilution of the stocks (see earlier discussion) with the prepared ‘‘clean’’water. It is also necessary to make a fresh solution of 4% paraformalde-hyde in 1� PBS. Several methods are available for preparing this solution,but I prefer the following: Put 300 ml of 1� PBS in an RNase-free flaskwith a treated stir bar and bring to an active boil in the microwave.Immediately place the flask on a magnetic stir plate under a fume hood

[32] gene expression techniques in plants 629

and add 12 g of paraformaldehyde to the hot solution. Stir. The powdershould go into solution quickly. Allow the flask to cool and cover themouth with parafilm or foil. Place the flask on ice under the hood to coolcompletely. This must be done before starting the prehybridization to allowthe solution to cool but should not be performed a day in advance.

The following is the prehybridization protocol. Unless otherwisenoted, the steps are performed in square polyethylene boxes in volumesof 300 ml.

1. Place slides into a metal slide rack, leaving every other slot emptyto allow circulation of the solutions.

2. Incubate 10 min in about 300 ml of Citrisolv in a glass staining box.Repeat with fresh Citrisolv.

3. Rehydrate the slides by processing through an ethanol series asfollows: 2 min in 100% ethanol, 1 min in 100%, and 1 min each in 95%,85%, 70%, 50%, and 30%. Follow by 2 min in 150 mM NaCl, and then2 min in 1� PBS.

4. Incubate the slides for 20 min at 37� in 1� pronase buffer (50 mMTris pH 7.5, 5 mM EDTA) with 10 �g/ml pronase.

Note on pronase treatment: Either pronase or proteinase K can be usedfor digestion, but they use different buffers, so care must be taken to ensureuse of the correct buffer. This step requires considerable optimization.First, the activities of both enzymes vary between preparations. Whenpreparing the enzyme stock, follow the manufacturer’s instructions to makea large volume (10–20 ml) at 10 mg/ml in DEPC water. This can be ali-quoted into small working volumes in RNase-free microtubes, which can bestored long term at �20�. This will allow the use of the same stock (andhence, the same activity level) throughout repeated experiments. Second,once the stock is prepared, the specific activity must be assessed forthe particular tissue being analyzed. The best way to do this is to run testdigestions on sectioned tissue using a range of concentrations (5–20 �g/ml)or incubation lengths (20–30 min). For simplicity, vary only one component(concentration, time, or temperature) in the experiment. Ideally, the tissueshould be digested as much as possible without affecting tissue/cell integri-ty. It is important to ensure that the cell contents are not affected by thedigestion (the cell walls may look perfect even when the contents are gone).The use of stains can aid in the evaluation of the optimal enzyme treatment.Different tissue types or stages may require different treatments. Thefindings of this process will determine the enzyme concentration that willbe used at this step in the actual prehybridization.

5. Following digestion, incubate in 1� PBS with 0.2% glycine for2 min.

630 functional analyses [32]

6. Rinse in 1� PBS for 2 min.7. Incubate in 4% formaldehyde for 20 min under a fume hood.8. Rinse in 1� PBS for 2 min (dispose of the PBS from this step in the

formaldehyde waste).9. Acetylation: Incubate slides for 10 min in 0.1 M triethanolamine

pH 8.0 with 0.5% acetic anhydride.

Note: This solution must be prepared fresh but takes a while to com-plete because the triethanolamine is a thick liquid and is difficult to pipette.It is best to start preparing the 0.1 M of triethanolamine during the incu-bation period of step 7. Under a fume hood, place a glass staining dish on amagnetic stir plate. Place a stir bar and two pieces of plastic sterile 10-mlpipettes in the bottom. The pipettes should be broken so that they fit in thebottom of the container with room for the stir bar to spin. Fill the dish with589.8 ml of ‘‘clean’’ water and add 7.8 ml of triethanolamine while stirring.Bring the pH to 8.0 with 2.4 ml of concentrated HCl. Check the pH using apH strip. Carefully add 3.6 ml of acetic anhydride. Momentarily stop thestir bar and allow the pipette pieces to settle on either side of the stir bar.Place the slide rack into the glass box so that the rack is supported abovethe stir bar with sufficient room for the bar to move. Restart the stirringand incubate for 10 min.

10. Rinse in 1� PBS for 2 min (do this step in the fume hood anddispose of the PBS in the acetic anhydride waste).

11. Rinse in 150 mM NaCl for 2 min. Follow by processing through theethanol dehydration series with 1 min of incubation per step: 30%, 50%,70%, 85%, 95%, and 100%. It is not necessary to make a fresh dehydrationseries; in fact, the same solutions can be used for several experiments.

12. Incubate in fresh 100% ethanol for 2 min. Carefully remove slidesfrom rack and place them section-side up onto fresh paper towel. Allow todry completely.

13. Prewarm the necessary number of tubes of hybridization solution ina heat block at 80� (this should correspond to the number of slide pairs, seestep 14). Set the hybridization oven to the appropriate temperature(see below).

14. Carefully examine the dry slides and choose those that have thebest quality of tissue at the desired stage. Arrange the chosen slides intopairs. Mark the pairs as to which probe will be used on them (each pair willhave the same probe). At least one pair should be hybridized with senseprobe as a negative control.

15. Probe should be used at 0.5 ng/�l/kb. The amount of probe perslide pair is determined by Lp � 100 �l � (0.5 ng/�l/kb), where Lp is thelength of the probe in kilobases. For instance, a probe hydrolyzed to

[32] gene expression techniques in plants 631

0.15 kb will require 0.15 � 100 � 0.5 ¼ 7.5 ng of probe. The concentrationof the probe is determined in step 12 in the section ‘‘Preparation of RNAProbes.’’ The first time that a given probe is hybridized, it should be tried atboth the concentration calculated above (this is the 1� concentration) andat 5�.

16. For each slide pair, the desired amount of probe should be added to50% deionized formamide to make the volume up to 40 �l. Heat the probeto 80�.

17. Add 200 �l of preheated hybridization solution to each tube ofheated probe and mix by pipetting. The hybridization solution is veryviscous and is difficult to pipette accurately.

18. Apply the probe to each slide pair. The technique of probe applica-tion is a matter of taste and requires some practice to determine what worksbest for you. This is the method I use: Take the first slide of the pair andpipette 100 �l of probe mix onto the slide. Carefully use the side of anRNase-free pipette tip to spread the hybridization solution across the entiresurface of the slide. Pipette the remaining probe solution onto the secondslide along the narrow edge of the slide, opposite the frosted area. Take thespread slide and place its corresponding edge on the narrow edge of thesecond slide. The wet side of the first slide should be facing with the sectionsdownward toward the second slide. Slowly, lower the spread slide onto thesecond slide, allowing the adhesion of the probe solution to pull the liquidacross the entire surface of the slide. The final product will be a slide‘‘sandwich’’ with the probe solution in between the surfaces of the two slides.

19. Elevate the slide sandwiches above wet paper towels in a flat plasticbox. This can be achieved by breaking plastic sterile 1-ml pipettes intohalves and placing them across the bottom of the box, which is lined withwet paper towels. Line the lid with Saran wrap and close tightly. Place thebox into the preheated hybridization oven for 14–16 h. Hybridizationtemperatures generally range from 38 to 45�. Initial hybridizations shouldbe performed at a lower temperature, such as 40�.

Make careful notes concerning the probes used and their concentration,the hybridization temperature, and any other variables such as the pronaseconcentration.

Posthybridization

At the end of the prehybridization protocol, several solutions for theposthybridization should be prepared 1 day in advance. Prepare 1.5 L of0.2� SSC and place at 48–55�. The temperature depends on the desiredstringency of your wash. A good rule of thumb is to wash at a temperature 10�

632 functional analyses [32]

above the hybridization temperature. Prepare 1.5 L of 1� NTE solutionand place at 37�. In addition, you can prepare 300 ml of 1� PBS; 650 ml of100 mM Tris 7.5, 150 mM NaCl; and 250 ml 100 mM Tris pH 9.5, 100 mMNaCl, 50 mM MgCl2. The morning of the posthybridization, severaladditional solutions should be prepared using the solutions above: 120 ml1.0% Roche Blocking Reagent (Roche 1 096 176) in 100 mM Tris 7.5,150 mM NaCl (this requires warming for complete incorporation, so allowtime for cooling before use), and 520 ml 1.0% BSA in 100 mM Tris 7.5,150 mM NaCl, and 0.3% Triton. The following is the posthybridizationprotocol. Unless otherwise noted, the incubations are performed in squarepolyethylene boxes in volumes of 300 ml.

1. Fill a square plastic box with about 300 ml of preheated 0.2� SSC.Immerse each slide sandwich and gently separate by opening, not sliding.Place the slides in a slide rack, leaving an empty slot between each one forcirculation.

2. Wash the slides for 1 h in fresh preheated 0.2� SSC with gentleagitation in a hybridization oven set to the chosen wash temperature.

3. Repeat 60-min wash with fresh preheated 0.2� SSC.4. Incubate the slides in preheated 1� NTE for 5 min at 37� with

gentle agitation.5. Repeat 5-min 1� NTE wash.6. Treat with 20 �g/ml RNase A in preheated 1� NTE for 20 min

at 37�.7. Incubate the slides in preheated 1� NTE for 5 min at 37� with

gentle agitation.8. Repeat 5-min 1� NTE wash.9. Wash for 60 min in preheated 0.2� SSC at wash temperature with

gentle agitation.10. Incubate 5 min in 1� PBS at RT.11. Place each slide on the bottom of a flat plastic box, section-side up

with no slides overlapping each other. The flat boxes cited above will hold10 slides. Fill each box with 100 ml of 1.0% Roche Blocking Reagent in100 mM Tris 7.5, 150 mM NaCl. This should be just enough to cover theslides. Incubate for 45 min on a rocking platform at RT.

12. Replace block solution with 100 ml 1.0% BSA in 100 mM Tris 7.5,150 mM NaCl, 0.3% Triton. Incubate for 45 min on a rocking platformat RT.

13. Dilute 8 �l of alkaline phosphatase-conjugated anti-DIG antibody(Roche 1 093 274) into 10 ml of 1.0% BSA in 100 mM Tris 7.5, 150 mMNaCl, 0.3% Triton. Make a puddle of the solution in a large plastic weighdish. Place each slide pair in the puddle with their long edges in the

[32] gene expression techniques in plants 633

solution. Sandwich the slides together, with the sections on the inside,drawing up the antibody solution between them. Drain the slide pair on astack of Kim wipes and place the edge back into the puddle. Solution willbe drawn up by capillary action. Repeat once, avoiding air bubbles.

14. Elevate the slide sandwiches with antibody solution above wetpaper towels as described above. Allow to sit at RT for 2 h.

15. Drain slides on Kim wipes and separate carefully. Place on thebottom of a flat plastic box as in step 11. Wash with 100 ml of 1.0% BSAin 100 mM Tris 7.5, 150 mM NaCl, 0.3% Triton for 15 min at RT on arocking platform. Repeat three times for a total of 4 washes.

16. Replace BSA/Triton solution with 100 ml 100 mM Tris pH 9.5,100 mM NaCl, 50 mM MgCl2. Wash for 10 min with rocking.

17. Fill a Coplin jar with 100 mM Tris pH 9.5, 100 mM NaCl, and50 mM MgCl2 and dip each slide into the solution to ensure that all ofthe detergent is removed.

18. Prepare substrate solution for color detection by adding 22 �l NBT(Roche 1 383 213) and 16 �l BCIP (Roche 1 383 221) to 10 ml of 100 mMTris pH 9.5, 100 mM NaCl, 50 mM MgCl2.

19. Apply solution to slide sandwiches as described in step 13.20. Elevate slide sandwiches above wet paper towels and seal box

tightly. Carefully wrap box in aluminum foil and place in a dark drawerto prevent light contamination.

Again, make notes on all the variables, such as the wash temperature.

Imaging

Color development generally takes 12–48 h. Good signal is usuallyvisible to the naked eye, although this depends on the size of the expressiondomain. Carefully examine the sandwiches under a microscope to assessstaining. When adequate signal is detectable, immerse the sandwiches inTE to separate and stop the staining reaction. Place slides into a slide rackand incubate in 1� PBS for 5 min at RT. Counterstaining with calcofluor(also known as Fluorescent Brightener, Sigma F-3397) can significantlyimprove contrast and visualization. Incubate slides in 0.002% solution ofcalcofluor in 1� PBS. Follow by rinsing for 5 min in 1� PBS. Dehydrationwill significantly reduce staining intensity, so mount slides with water,glycerol, or other aqueous medium (I prefer water). Do not let the slidesdry out at any step.

The slides can now be photographed. To take advantage of the calco-fluor counterstaining, sections should be illuminated simultaneously withfluorescent and white light. Dial down the intensity of the white light untilthe fluorescence of the tissue is just visible. This will appear as a blue-white

634 functional analyses [32]

glow in the cell walls. Certain tissue absorbs calcofluor better than othersand this may be inconsistent between sections. ‘‘Real’’ signal should belight brown or lilac to dark brown. Dark blue staining is generally not realsignal (see below). Photodocument every section with informative stainingbecause water-mounted slides will not be stable for more than a week ortwo (at 4� elevated above wet paper towels).

Troubleshooting In Situ Hybridization Results

As mentioned earlier, performing in situ hybridization can be a chal-lenging process and the protocol requires optimization of many steps. Thefollowing are suggestions for troubleshooting:

No or low signal: This may be due to a number of factors includinglow probe concentration, hybridization, or wash temperaturesthat are too high, RNase contamination of reagents (particularlyhybridization solution), overfixation of tissue, insufficient tissuedigestion, or improper probe hydrolization (see below).

High background: This typically results from hybridization or washtemperatures that are too low. It is important to note that highbackground can be general, affecting all tissues, or disconcertinglyspecific, only affecting certain tissues. Distinguishing nonspecificstaining from ‘‘real’’ staining is sometimes difficult. Tissues thatare prone to nonspecific staining include stamens and pollen, vas-cular tissues, and very small dense meristematic cells. Nonspecificsignal often takes the form of very dark blue staining that isassociated with the cell walls. It is important to remember thatin situ hybridization targets mRNA, which should be in the cyto-plasm, not the nucleus or cell walls. The caveat to this statement isthat in mature plant cells that are highly vacuolated, the cytoplasmmay be closely pressed to the cell wall. The most commonly ana-lyzed genes for comparative gene expression, such as MADS-boxcontaining genes or CYCLOIDIA homologues, typically have highlevels of expression that should be clearly discernible. Comparisonswith sense control slides can sometimes help to distinguish the realsignal from the nonspecific staining.

Tissue appears to be degraded or cell contents are gone: Tissue hasbeen overdigested. Lower the enzyme concentration.

Note on probe hydrolysis: Protocols commonly call for probe to behydrolyzed to 70–150 bp (Jackson, 1991; Weigel and Glazebrook,2002). Although this works well for many probes, others performbetter at different lengths. Optimization of probe length is a trial-by-error process; there are no good rules of thumb. Although 200 bp

[32] gene expression techniques in plants 635

may work well for a gene in one species, the ortholog in anothertaxon may work best at 300 bp. If little signal is observed even at lowhybridization temperatures and high probe concentrations, using ashorter probe is often successful. Alternatively, high background(either general or specific) can be eliminated by using longer probe.

Several alternatives to the type of in situ hybridization described earlierhave been developed. For organs with complex three-dimensional struc-ture, whole-mount in situ hybridization (Zachgo et al., 2000) can yield moreinformative results. Another alternative that has yet to be broadly used isin situ RT-PCR. One drawback to the DIG-labeled form of this protocol(Johansen, 1997) is that it sometimes results in nonspecific labeling of allnuclei, which can make interpretation of the data difficult. A new methodusing Oregon green–labeled UTP (Ruiz-Medrano et al., 1999) has yieldedbeautiful results, however (Kim et al., 2003a,b).

Concluding Remarks

The generation of gene expression data is only the first step in thedifficult process of assessing gene function. It has been demonstrated formany genes that expression is not necessarily a simple proxy for the spatialextent of gene function, both because of non–cell autonomous effects andposttranscriptional or posttranslational regulation. Therefore, any dataresulting from the types of analyses described earlier should be interpretedwith care. However, given that direct analyses of gene function usinggenetic tools are commonly unavailable in nonmodel species, gene expres-sion is often our only comparative tool. Therefore, it is preferable to use acombination of all of the aforementioned techniques to obtain a detailedand clear picture of gene expression patterns.

References

Chang, S., Puryear, J., and Cairney, J. (1993). A simple and efficient method for isolating

RNA from pine trees. Plant Mol. Biol. Rep. 11, 113–116.

Jackson, D. (1991). In situ hybridisation in plants. In ‘‘Molecular Plant Pathology: A Practical

Approach’’ (D. J. Bowles, S. J. Gurr, and P. McPherson, eds.), pp. 163–174. Oxford

University Press, Oxford.

Johansen, B. (1997). In situ PCR on plant material with sub-cellular localization. Ann. Bot. 80,

697–700.

Kim, M., McCormick, S., Timmermans, M., and Sinha, N. (2003a). The expression domain of

PHANTASTICA determines leaflet placement in compound leaves. Nature 424, 438–443.

Kim, M., Pham, T., Hamidi, A., McCormick, S., Kuzoff, R. K., and Sinha, N. (2003b).

Reduced leaf complexity in tomato wiry mutants suggests a role for PHAN and KNOX

genes in generating compound leaves. Development 130, 4405–4415.

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Kramer, E. M., Dorit, R. L., and Irish, V. F. (1998). Molecular evolution of genes controlling

petal and stamen development: Duplication and divergence within the APETALA3 and

PISTILLATA MADS-box gene lineages. Genetics 149, 765–783.

Ruiz-Medrano, R., Xoconostle-Cazares, B., and Lucsa, W. (1999). Phloem long-distance

transport of CmNACP mRNA: Implications for supracellular regulation in plants.

Development 126, 4405–4419.

Sambrook, J., and Russel, D. W. (2001a). ‘‘Molecular Cloning,’’ Vol. 2. Cold Spring Harbor

Press, Cold Spring Harbor, NY.

Sambrook, J., and Russel, D. W. (2001b). ‘‘Molecular Cloning,’’ Vol. 3. Cold Spring Harbor

Press, Cold Spring Harbor, NY.

Suzuki, Y., Hibino, T., Kawazu, T., Wada, T., Kihara, T., and Koyama, H. (2003). Extraction

of total RNA from leaves of Eucalyptus and other woody and herbaceous plants using

sodium isoascorbate. Biotechniques 34, 988–993.

Wang, S. X., Hunter, W., and Plant, A. (2000). Isolation and purification of functional total

RNA form woody branches and needles of Sitka and white spruce. Biotechniques 28,

292–296.

Weigel, D., and Glazebrook, J. (2002). ‘‘Arabidopsis: A Laboratory Manual.’’ Cold Spring

Harbor Press, Cold Spring Harbor, NY.

Zachgo, S., Perbal, M.-C., Saedler, H., and Schwarz-Sommer, Z. (2000). In situ analysis of

RNA and protein expression in whole mounts facilitates detection of floral gene

expression dynamics. Plant J. 23, 697–702.

[33] Developing Antibodies to Synthetic PeptidesBased on Comparative DNA Sequencing of

Multigene Families

By Roger H. Sawyer, Travis C. Glenn, Jeffrey O. French, andLoren W. Knapp

Abstract

Using antisera to analyze the expression of specific gene products is acommon procedure. However, in multigene families, such as the �-keratinsof the avian integument where strong homology exists among the scale(Sc�K), claw (Cl�K), feather (F�K), and feather-like (Fl�K) subfamilies,determining the cellular and tissue expression patterns of the subfamilies isdifficult because polyclonal antisera produced from any one protein recog-nize all family members. Traditionally, researchers produced and screenedmultiple monoclonal antisera produced from the proteins of interest untilan antiserum with sufficient specificity could be obtained. Unfortunately,this approach requires a lot of effort, and once obtained, such antisera mayhave limited applications. Here, we present procedures by which compara-tive DNA sequences of members from the �-keratin multigene family were

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