- Email: [email protected]
[49] Tissue-print hybridization for detecting RNA directly
688
OTHER METHODS
[49]
carefully blotted or removed from the nitrocellulose filter. Special attention must be taken when drying tissue sections that have high water content, such as developing shoots. This consideration will eliminate the transfer of soluble material from the tissue section outside the print made on the nitrocellulose membrane. 2. The detection of alkaline phosphatase-conjugated second antibody on tissue prints was selected over the peroxidase-conjugated second antibody procedure because the substrates used for detecting the peroxidase, such as o-phenylenediamine and H202, are capable of detecting endogenous peroxidase activity in plant tissue sections. 33 Thus the endogenous peroxidase activity in the tissue plant will mask the immunoblotting reaction. Conclusions As new structural cell wall proteins are discovered, it is likely that preparation of specific antibodies for these proteins will continue to provide useful information on how different cell walls are constructed. The use of the tissue-print Western blot technique with a new set of cell wall antibodies will be useful for screening many plant tissues and plant species because it is a simple immunolocalization procedure. Acknowledgment I am grateful to Dr. Joseph E. Varner for support and for reviewingthe manuscript.
[49] T i s s u e - P r i n t H y b r i d i z a t i o n for D e t e c t i n g R N A D i r e c t l y
By
TOM
J.
GUILFOYLE,
BRUCE
A.
GRETCHEN
MCCLURE,
MELISSA
A.
GEE,
and
HAGEN
Varner et al.l first described tissue-print hybridization with plant organs by employing a modification of an immunological tissue-printing procedure developed by Cassab and Varner. 2 McClure and Guilfoyle3'4 modified the hybridization procedure for detecting moderately abundant t j. E. Varner, Y.-R. Song, L.-S. Lin, and H. Yuen, in " T h e Molecular Basis of Plant D e v e l o p m e n t " (R. Goldberg, ed.), p. 161. Alan R. Liss, N e w York, 1989. 2 G. I. C a s s a b and J. E. Varner, J. Cell Biol. 105, 2581 (1989). 3 B. A. McClure and T. J. Guilfoyle, Science 243, 91 (1989). 4 B. A. McClure and T. J. Guilfoyle, Plant Mol. Biol. 12, 517 (1989).
METHODS IN ENZYMOLOGY,VOL. 218
Copyright © 1993by AcademicPress, Inc. All rights of reproduction in any form reserved.
[49]
TISSUE-PRINT HYBRIDIZATION FOR DETECTING R N A
689
mRNA transcripts with 35S-labeled antisense RNA probes. Subsequently, other modifications of these procedures have been reported by Mansky et al. 5
Varner et al. l originally used tissue prints and 32p-labeled cDNA probes to detect extensin mRNA in soybean pods and fl-conglycinin a-subunit mRNA in developing soybean seeds. Tissue-print hybridization with 35Slabeled antisense RNA probes was used to detect the organ and tissue distribution of auxin-responsive mRNAs in whole seedlings and organ sections 3'4'6 and in hypocotyl and epicotyl sections undergoing gravitropic curvature? Ye and Varner 7 have localized the expression of mRNAs that encode hydroxyproline-rich glycoproteins (HRGPs) and glycine-rich proteins (GRPs) in developing soybean tissues using tissue print and in situ hybridization with 35S-labeled antisense RNA probes. Principle Tissue printing is a simple method for detecting macromolecules blotted directly from the surfaces of severed organs onto nylon or nitrocellulose membranes. The blotting procedure produces an image of the cut surface of the tissues on the membrane, and macromolecules such as proteins, complex carbohydrates, and nucleic acids are fixed to the membrane. The retention of nucleic acids on the membrane allows the detection of RNAs by hybridization with either DNA or antisense RNA probes.
Materials Reagents
Nylon membranes (Zeta-Probe; Bio-Rad, Richmond, CA) Whatman 3MM paper (Fisher Scientific, St. Louis, MO) Kimwipes (Kimberly-Clark Corp., Roswell, GA) Ultraviolet (UV) light sources (260 and 300-320 nm) Kapak/Scotchpak pouches (Kapak Corporation, Bloomington, MN) Phenol : chloroform : isoamyl alcohol (25 : 24 : 1) T7 RNA polymerase and vectors (GIBCO-Bethesda Research Laboratories, Gaithersburg, MD) or any one of a number of kits available commercially for synthesizing antisense or sense RNA probes (Promega, Madison, WI; Stratagene, La Jolla, CA) 5 L. M. Mansky, R. E. Andrews, Jr., D. P. Durand, and J. H. Hill, Plant Mol. Biol. Rep. 8, 13 (1990). 6 A. R. Franco, M. A. Gee, and T. J. Guilfoyle, J. Biol. Chem. 265, 15845 (1990). 7 Z.-H. Ye and J. E. Varner, Plant Cell. 3, 23 (1991).
690
OTHER METHODS
[49]
Placental ribonuclease inhibitor and RNase-free DNase (Promega) ATP, CTP, GTP, dithiothreitol (DTT), sodium dodecyl sulfate (SDS), salmon sperm DNA, poly(A), yeast tRNA, polyvinylpyrrolidone, bovine serum albumin, Ficoll, and formamide (Sigma Chemical Company, St. Louis, MO): The formamide is deionized prior to use by stirring for 30 rain in AG501-X8 ion-exchange resin (Bio-Rad) as described by Sambrook et al. 8 [35S]Thio-UTP (> 1200 Ci/mmol) (NEG039; New England Nuclear, Boston, MA) Sephadex G-50 (Pharmacia, LKB Biotechnology, Inc., Piscataway, N J) India ink (Higgins No. 4415; Faber-Castell Corporation, Newark, NJ) Kodak XRP-5 X-ray film, Tmax 400, Tech Pan 2415, and Tech Pan 4415 photographic films, Kodak Tmax and HC-110 developer, stop bath, and rapid fix (Eastman Kodak, Rochester, NY) Solutions
SSC (10×): 1.5 M NaCI, 0.15 M sodium citrate SSPE (10×): 1.8 M NaCi, 0.1 M sodium phosphate (pH 7.4), 0.01 M ethylenediaminetetraacetic acid (EDTA) Denhardt's solution (50 ×): 5 g Ficoll, 5 g polyvinylpyrrolidone, and 5 g bovine serum albumin (BSA) brought to 500 ml with H20
Methods M e m b r a n e s a n d Printing T e c h n i q u e
For tissue printing, a dry nylon membrane is placed over a single layer of dry Whatman 3MM paper or some other absorbent paper. Vinyl medical gloves should be worn when handling the nylon membranes and when blotting the tissue sections to the membranes to prevent the transfer of fingerprints to the membrane, which can prevent proper wetting of the membrane. Organs or organ sections are prepared for printing onto membranes by sectioning through the organ with a single- or double-edged razor blade. 9 The freshly cut surfaces are pressed immediately to the nylon membrane or lightly blotted with Kimwipes prior to blotting to the membrane (depending on the moisture content of the tissue). Tissue printing is performed by using firm pressure with the index finger above the sectioned organ for 8j. Sambrook,E. F. Fritsch,and T. Maniatis, "MolecularCloning:A LaboratoryManual." Cold Spring Harbor Press, Cold Spring Harbor, New York, 1989. 9 B. A. McClure and T. J. Guilfoyle,Plant Mol. Biol. 9, 611 (1987).
[49]
TISSUE-PRINT HYBRIDIZATION FOR DETECTING R N A
691
30 to 120 sec. In some cases, we have used a thin piece of flexible cardboard to c o v e r the tissue sections and, by applying a relatively uniform pressure above the cardboard, multiple or larger tissue sections can be printed at the same time. The amount of pressure applied should not be excessive, but should be sufficient to imprint an image of the section on the membrane. After printing, the organ sections can be removed from the membrane with the aid of a forceps or spatula. The nylon membrane is then allowed to dry at room temperature. We evaluate the quality of the tissue prints by examining the printed nylon membrane under a 300- to 320-nm UV light source. Under UV light, it is possible to observe whether any organ sections were crushed or distorted during blotting. U n e v e n blots may result from too much or too little finger pressure. Distortions of prints may result from uneven pressure over the section or movement of the section on the membrane during printing. Each type of organ or tissue has a characteristic consistency, turgidity, and cellular architecture and, therefore, the amount of pressure required to obtain even, consistent prints must be experimentally determined. In our experience, large organs of firm consistency such as coytledons, stems, and petioles are much easier to tissue print than small or less firm organs such as roots, leaves, or floral parts. We have kept dried prints at room temperature for several weeks, but it is best to use the prints immediately or store them at 4 ° in sealed Kapak/ Scotchpak pouches until the prints are used for hybridization. Preparation o f Antisense and Sense R N A Probes A number of suitable vectors with T7, T3, or SP6 promoters that flank multiple cloning sites for inserting full-length or partial-length cDNAs are commercially available (GIBCO-Bethesda Research Laboratories, Promega, Stratagene). To generate RNA probes, full-length or partial-length cDNAs are cloned into transcription vectors, and R N A is synthesized in vitro with the appropriate R N A polymerase and transcription buffer. Prior to carrying out the R N A polymerase reaction, the D N A template is linearized by restriction e n z y m e cleavage. The linearization of the D N A template should be checked by agarose gel electrophoresis. 8 After confirmation of linearity, the D N A is extracted with 1 vol of phenol : chloroform : isoamyl alcohol (25 : 24 : 1), and precipitated from the aqueous phase with 2 vol of 95% (v/v) ethanol at - 80 ° for 1 hr. The ethanol-precipitated DNA is recovered by centrifugation in a microfuge at top speed for 15 min. After removal of the supernatant, the DNA is dried in vacuo. The dried D N A pellet is suspended in sterile, deionized water. The concentra-
692
OTHERMETHODS
[49]
tion of the DNA can be determined by absorbance at 260 nm with a UV spectrophotometer. The antisense or sense RNA is synthesized according to the instructions provided by the vendor that supplies the DNA template vector and RNA polymerase. We have used the following protocol for synthesis of RNA probes. Reactions are carried out in a 10-/xl mixture containing T7 RNA polymerase buffer [40 mM Tris-HCl (pH 7.9), 6 mM MgC12, 10 mM dithiothreitol, 2 mM spermidine], 0.3 to 0.7/~g of linearized DNA template, 10 units (U) or 1 t~l of T7 RNA polymerase, 30-40 U of placental ribonuclease inhibitor, ATP, CTP, and GTP (1 mM each), and 0.1 to 0.5 mCi of [35S]thio-UTP. We incubate reaction mixtures at 37° for 40-60 min, and then add a second aliquot of T7 RNA polymerase (10 U) and incubation is continued for an additional 40-60 min. After the incubation period, the DNA template is removed by adding RNase-free DNase I (I U) to the reaction mixture and incubating for an additional 15 min at 37 °. Unincorporated nucleotides are removed from the RNA transcript by passing the reaction mixture through a 0.5-ml spun column of Sephadex G-50. 8 Although we routinely use [35S]thio-UTP, [o~-32p]UTP or any other 32p-labeled ribonucleoside triphosphate can be used to synthesize the antisense or sense RNA probe. The 32p-labeled probes allow shorter exposure times and are less expensive to synthesize, but provide less autoradiographic resolution compared to the 35S-labeled probes. Although we generally use probes of high specific activity, the amount and specific activity of32p - or 35S-labeled ribonucleotide used to synthesize the RNA probe can be altered depending on how rapidly one wants to detect a hybridization signal and how abundant the mRNA is within the tissue section.
Hybridization o f R N A to Tissue Print Prior to hybridization, dried tissue prints should be washed for 4-12 hr in 0.1-0.2 x SSC containing 1% (w/v) SDS at 65°. After this washing step, prehybridization and hybridization are carried out in 1.5 x SSPE, 1% (w/v) SDS, 1% (w/v) nonfat powdered milk, 0.5 mg/ml denatured, sonicated salmon DNA, and 100 mM dithiothreitol at 68 °. We generally carry out prehybridization of the tissue print membranes for 12-16 hr, and follow this by hybridization with the antisense or sense RNA probes at 5 × 10 7 counts per minute (cpm)/ml in fresh buffer for 12-24 hr. After hybridization, tissue prints should be rinsed briefly in 2 x SSC, 1% SDS, and 10 mM dithiothreitol, and then washed two more times in fresh changes of 2 × SSC, I% SDS, and 10 mM dithiothreitol for 30 min each at 42 ° with gentle shaking. Two additional washes should be carried
[49]
TISSUE-PRINT HYBRIDIZATION FOR DETECTING R N A
693
out in 0.2 x SSC, 1% SDS, and 1 mM dithiothreitol at 65° for 30 min each. The membrane can then be dried and analyzed by autoradiography. As an alternative to the prehybridization and hybridization buffer described above, we have substituted a buffer containing 50% (v/v) formamide, 5 x Denhardt's solution, 8 6x SSC, 2 mM EDTA, 0.1% SDS, 200/.~g/ml poly(A), 100/~g/ml yeast tRNA, and 70 mM dithiothreitol. With this buffer system, prehybridization and hybridization should be carried out at 42° . Filters should be washed as described above following hybridization.
Staining Procedure Tissue prints can be stained with India ink2 or other dyes before autoradiography. Before staining, tissue prints should be briefly rinsed in icecold water and then immersed in ice-cold India ink (Higgins No. 4415; Faber-Castell Corporation) for 1-10 rain. Tissue prints can be destained by briefly rinsing in ice water and then by rinsing several times in 0.2 x SSC and 1% SDS. Although we have found that the tissue prints are not always uniformly stained with India ink, the stained images, nevertheless, provide a useful comparison to the autoradiographic images. The ink-stained images reveal anatomical detail that is not obvious in the autoradiograms, and provide an image for better interpretation of localized mRNA expression patterns on the autoradiograms. The ink-stained images are also useful for interpreting autoradiograms in terms of ineffective, incomplete, or distorted blotting of the sections onto the membrane.
Autoradiography Procedure We use Kodak XRP-5 film exposed for 24-48 hr at - 7 0 ° to evaluate initially the quality and quantity of hybridization to the tissue prints. Although exposure of the prints on XRP-5 is suitable for some tissue prints, autoradiograms of higher resolution can be obtained by exposing the tissue prints to the photographic film, Tmax 400. Exposure times on Tmax 400 are about five times longer than tissue prints exposed on XRP-5, but a higher quality image is obtained with the Tmax 400. With SAUR (small auxin up-regulated RNA) 4probes shown in Fig. 1, the autoradiograms on Tmax 400 were exposed for about 10 days at - 7 0 °. We develop the film for 6-11 min at 24° in Kodak Tmax developer. This is followed by Kodak stop bath for 30 sec and Kodak rapid fix for 5 min. The film is then washed in running tap water for 5 min, rinsed in Kodak, Photo-Flo 200, and air dried.
694
OTHER METHODS
[49]
FIG. 1. Tissue-print hybridization reveals gravity-responsive mRNAs in soybean seedlings. Soybean seedlings were grown in the normal, vertical orientation or placed in a horizontal orientation for 20 rain prior to tissue planting. The reorientation of the soybean seedlings from the vertical to the horizontal position results in negative gravitropic curvature 20-45 min after the seedlings are placed in the horizontal position. Auxin-responsivernRNAs are detected in the epidermis and cortex tissues of the elongating region of the vertical hypocotyl with an SAUR antisense RNA hybridization probe labeled with [35S]thio-UTP? Tissue-print hybridization shows that the SAUR mRNAs disappear from the top of the horizontal hypocotyl prior to gravitropic bending. Schematic representations of the soybean seedlings are shown to the left or above the autoradiograms.
We have tested a number of other films for exposures of tissue-print hybridizations. K o d a k Tri-X Pan does not perform as well as Tmax 400, and exposure time for K o d a k Tmax 3200 is not substantially faster than Tmax 400. We have obtained the highest quality images with K o d a k Tech Pan 2415 (35-mm format) or K o d a k Tech Pan 4415 (4 × 5 in. format). The film speeds o f Tech Pan 2415 and 4415 film are considerably slower than Tmax 400. Tech Pan films have a fine grain and produce high-quality autoradiograms of tissue prints, but the slow speed of these films limits their application to tissue prints that have strong hybridization signals.
Comments Tissue-print hybridization provides an alternative method to in situ hybridization with fixed tissue sections. Tissue-print hybridization provides a reliable method to detect organ-specific and tissue-specific gene
[50]
CLONING FROM DRIED AGAROSE GELS
695
expression because the patterns of hybridization observed are similar, if not identical, to those observed with in situ hybridization. 3'5'6'1° Tissueprint hybridization is much less time consuming and less expensive than in situ hybridization, and requires a minimal amount of technical expertise and equipment. Tissue-print hybridization also has the advantage that numerous tissue treatments or manipulations can be examined with a minimal amount of effort. For example, all of the tissue sections can be printed on a single piece of nylon membrane and, once the tissue prints have been made for each treatment or manipulation, staining, prehybridization, hybridization, and autoradiography can be uniformly carried out on that single nylon membrane. 10 M. A. Gee, G. Hagen, and T. J. Guilfoyle, Plant Cell 3, 419 (1991),
[50] R e c o v e r y a n d C l o n i n g o f G e n o m i c D N A F r a g m e n t s from Dried Agarose Gels By
MICHAEL W. MATHER,
J.
ANDREW KEIGHTLEY,
and JAMES A.
FEE
We describe here a method for the cloning of bacterial genes that circumvents the need to prepare and maintain genomic DNA libraries. Genomic DNA samples, highly enriched in specific restriction fragments, are isolated from dried agarose gels and used directly for cloning. 1 The method is rapid and technically simple, and reduces the possibility that a DNA sequence of interest might be missed due to underrepresentation in a library. Principle of the Method An analytical in-gel hybridization is employed to detect those genomic DNA restriction fragments that contain the sequence(s) of interest. An enriched fraction is subsequently isolated from a preparative-scale dried agarose gel, ligated into an appropriate vector, and propagated in Escherichia coli, Escherichia coil clones containing the fragment of interest are identified by a low-density colony hybridization screen. Hybridization in dried agarose gels was first described by Shinnick et al. a The technique is rapid, versatile, and sensitive. With radiolabeled l M. W. Mather, BioTechniques 6, 444 (1988). 2 T. M. Shinnick, E. Lurid, O. Smithies, and F. R. Blattner, Nucleic Acids Res. 2, 1911 (1975).
METHODS IN ENZYMOLOGY,VOL. 218
Copyright © 1993by Academic Press, Inc. All rights of reproductionin any form reserved.
OTHER METHODS
[49]
carefully blotted or removed from the nitrocellulose filter. Special attention must be taken when drying tissue sections that have high water content, such as developing shoots. This consideration will eliminate the transfer of soluble material from the tissue section outside the print made on the nitrocellulose membrane. 2. The detection of alkaline phosphatase-conjugated second antibody on tissue prints was selected over the peroxidase-conjugated second antibody procedure because the substrates used for detecting the peroxidase, such as o-phenylenediamine and H202, are capable of detecting endogenous peroxidase activity in plant tissue sections. 33 Thus the endogenous peroxidase activity in the tissue plant will mask the immunoblotting reaction. Conclusions As new structural cell wall proteins are discovered, it is likely that preparation of specific antibodies for these proteins will continue to provide useful information on how different cell walls are constructed. The use of the tissue-print Western blot technique with a new set of cell wall antibodies will be useful for screening many plant tissues and plant species because it is a simple immunolocalization procedure. Acknowledgment I am grateful to Dr. Joseph E. Varner for support and for reviewingthe manuscript.
[49] T i s s u e - P r i n t H y b r i d i z a t i o n for D e t e c t i n g R N A D i r e c t l y
By
TOM
J.
GUILFOYLE,
BRUCE
A.
GRETCHEN
MCCLURE,
MELISSA
A.
GEE,
and
HAGEN
Varner et al.l first described tissue-print hybridization with plant organs by employing a modification of an immunological tissue-printing procedure developed by Cassab and Varner. 2 McClure and Guilfoyle3'4 modified the hybridization procedure for detecting moderately abundant t j. E. Varner, Y.-R. Song, L.-S. Lin, and H. Yuen, in " T h e Molecular Basis of Plant D e v e l o p m e n t " (R. Goldberg, ed.), p. 161. Alan R. Liss, N e w York, 1989. 2 G. I. C a s s a b and J. E. Varner, J. Cell Biol. 105, 2581 (1989). 3 B. A. McClure and T. J. Guilfoyle, Science 243, 91 (1989). 4 B. A. McClure and T. J. Guilfoyle, Plant Mol. Biol. 12, 517 (1989).
METHODS IN ENZYMOLOGY,VOL. 218
Copyright © 1993by AcademicPress, Inc. All rights of reproduction in any form reserved.
[49]
TISSUE-PRINT HYBRIDIZATION FOR DETECTING R N A
689
mRNA transcripts with 35S-labeled antisense RNA probes. Subsequently, other modifications of these procedures have been reported by Mansky et al. 5
Varner et al. l originally used tissue prints and 32p-labeled cDNA probes to detect extensin mRNA in soybean pods and fl-conglycinin a-subunit mRNA in developing soybean seeds. Tissue-print hybridization with 35Slabeled antisense RNA probes was used to detect the organ and tissue distribution of auxin-responsive mRNAs in whole seedlings and organ sections 3'4'6 and in hypocotyl and epicotyl sections undergoing gravitropic curvature? Ye and Varner 7 have localized the expression of mRNAs that encode hydroxyproline-rich glycoproteins (HRGPs) and glycine-rich proteins (GRPs) in developing soybean tissues using tissue print and in situ hybridization with 35S-labeled antisense RNA probes. Principle Tissue printing is a simple method for detecting macromolecules blotted directly from the surfaces of severed organs onto nylon or nitrocellulose membranes. The blotting procedure produces an image of the cut surface of the tissues on the membrane, and macromolecules such as proteins, complex carbohydrates, and nucleic acids are fixed to the membrane. The retention of nucleic acids on the membrane allows the detection of RNAs by hybridization with either DNA or antisense RNA probes.
Materials Reagents
Nylon membranes (Zeta-Probe; Bio-Rad, Richmond, CA) Whatman 3MM paper (Fisher Scientific, St. Louis, MO) Kimwipes (Kimberly-Clark Corp., Roswell, GA) Ultraviolet (UV) light sources (260 and 300-320 nm) Kapak/Scotchpak pouches (Kapak Corporation, Bloomington, MN) Phenol : chloroform : isoamyl alcohol (25 : 24 : 1) T7 RNA polymerase and vectors (GIBCO-Bethesda Research Laboratories, Gaithersburg, MD) or any one of a number of kits available commercially for synthesizing antisense or sense RNA probes (Promega, Madison, WI; Stratagene, La Jolla, CA) 5 L. M. Mansky, R. E. Andrews, Jr., D. P. Durand, and J. H. Hill, Plant Mol. Biol. Rep. 8, 13 (1990). 6 A. R. Franco, M. A. Gee, and T. J. Guilfoyle, J. Biol. Chem. 265, 15845 (1990). 7 Z.-H. Ye and J. E. Varner, Plant Cell. 3, 23 (1991).
690
OTHER METHODS
[49]
Placental ribonuclease inhibitor and RNase-free DNase (Promega) ATP, CTP, GTP, dithiothreitol (DTT), sodium dodecyl sulfate (SDS), salmon sperm DNA, poly(A), yeast tRNA, polyvinylpyrrolidone, bovine serum albumin, Ficoll, and formamide (Sigma Chemical Company, St. Louis, MO): The formamide is deionized prior to use by stirring for 30 rain in AG501-X8 ion-exchange resin (Bio-Rad) as described by Sambrook et al. 8 [35S]Thio-UTP (> 1200 Ci/mmol) (NEG039; New England Nuclear, Boston, MA) Sephadex G-50 (Pharmacia, LKB Biotechnology, Inc., Piscataway, N J) India ink (Higgins No. 4415; Faber-Castell Corporation, Newark, NJ) Kodak XRP-5 X-ray film, Tmax 400, Tech Pan 2415, and Tech Pan 4415 photographic films, Kodak Tmax and HC-110 developer, stop bath, and rapid fix (Eastman Kodak, Rochester, NY) Solutions
SSC (10×): 1.5 M NaCI, 0.15 M sodium citrate SSPE (10×): 1.8 M NaCi, 0.1 M sodium phosphate (pH 7.4), 0.01 M ethylenediaminetetraacetic acid (EDTA) Denhardt's solution (50 ×): 5 g Ficoll, 5 g polyvinylpyrrolidone, and 5 g bovine serum albumin (BSA) brought to 500 ml with H20
Methods M e m b r a n e s a n d Printing T e c h n i q u e
For tissue printing, a dry nylon membrane is placed over a single layer of dry Whatman 3MM paper or some other absorbent paper. Vinyl medical gloves should be worn when handling the nylon membranes and when blotting the tissue sections to the membranes to prevent the transfer of fingerprints to the membrane, which can prevent proper wetting of the membrane. Organs or organ sections are prepared for printing onto membranes by sectioning through the organ with a single- or double-edged razor blade. 9 The freshly cut surfaces are pressed immediately to the nylon membrane or lightly blotted with Kimwipes prior to blotting to the membrane (depending on the moisture content of the tissue). Tissue printing is performed by using firm pressure with the index finger above the sectioned organ for 8j. Sambrook,E. F. Fritsch,and T. Maniatis, "MolecularCloning:A LaboratoryManual." Cold Spring Harbor Press, Cold Spring Harbor, New York, 1989. 9 B. A. McClure and T. J. Guilfoyle,Plant Mol. Biol. 9, 611 (1987).
[49]
TISSUE-PRINT HYBRIDIZATION FOR DETECTING R N A
691
30 to 120 sec. In some cases, we have used a thin piece of flexible cardboard to c o v e r the tissue sections and, by applying a relatively uniform pressure above the cardboard, multiple or larger tissue sections can be printed at the same time. The amount of pressure applied should not be excessive, but should be sufficient to imprint an image of the section on the membrane. After printing, the organ sections can be removed from the membrane with the aid of a forceps or spatula. The nylon membrane is then allowed to dry at room temperature. We evaluate the quality of the tissue prints by examining the printed nylon membrane under a 300- to 320-nm UV light source. Under UV light, it is possible to observe whether any organ sections were crushed or distorted during blotting. U n e v e n blots may result from too much or too little finger pressure. Distortions of prints may result from uneven pressure over the section or movement of the section on the membrane during printing. Each type of organ or tissue has a characteristic consistency, turgidity, and cellular architecture and, therefore, the amount of pressure required to obtain even, consistent prints must be experimentally determined. In our experience, large organs of firm consistency such as coytledons, stems, and petioles are much easier to tissue print than small or less firm organs such as roots, leaves, or floral parts. We have kept dried prints at room temperature for several weeks, but it is best to use the prints immediately or store them at 4 ° in sealed Kapak/ Scotchpak pouches until the prints are used for hybridization. Preparation o f Antisense and Sense R N A Probes A number of suitable vectors with T7, T3, or SP6 promoters that flank multiple cloning sites for inserting full-length or partial-length cDNAs are commercially available (GIBCO-Bethesda Research Laboratories, Promega, Stratagene). To generate RNA probes, full-length or partial-length cDNAs are cloned into transcription vectors, and R N A is synthesized in vitro with the appropriate R N A polymerase and transcription buffer. Prior to carrying out the R N A polymerase reaction, the D N A template is linearized by restriction e n z y m e cleavage. The linearization of the D N A template should be checked by agarose gel electrophoresis. 8 After confirmation of linearity, the D N A is extracted with 1 vol of phenol : chloroform : isoamyl alcohol (25 : 24 : 1), and precipitated from the aqueous phase with 2 vol of 95% (v/v) ethanol at - 80 ° for 1 hr. The ethanol-precipitated DNA is recovered by centrifugation in a microfuge at top speed for 15 min. After removal of the supernatant, the DNA is dried in vacuo. The dried D N A pellet is suspended in sterile, deionized water. The concentra-
692
OTHERMETHODS
[49]
tion of the DNA can be determined by absorbance at 260 nm with a UV spectrophotometer. The antisense or sense RNA is synthesized according to the instructions provided by the vendor that supplies the DNA template vector and RNA polymerase. We have used the following protocol for synthesis of RNA probes. Reactions are carried out in a 10-/xl mixture containing T7 RNA polymerase buffer [40 mM Tris-HCl (pH 7.9), 6 mM MgC12, 10 mM dithiothreitol, 2 mM spermidine], 0.3 to 0.7/~g of linearized DNA template, 10 units (U) or 1 t~l of T7 RNA polymerase, 30-40 U of placental ribonuclease inhibitor, ATP, CTP, and GTP (1 mM each), and 0.1 to 0.5 mCi of [35S]thio-UTP. We incubate reaction mixtures at 37° for 40-60 min, and then add a second aliquot of T7 RNA polymerase (10 U) and incubation is continued for an additional 40-60 min. After the incubation period, the DNA template is removed by adding RNase-free DNase I (I U) to the reaction mixture and incubating for an additional 15 min at 37 °. Unincorporated nucleotides are removed from the RNA transcript by passing the reaction mixture through a 0.5-ml spun column of Sephadex G-50. 8 Although we routinely use [35S]thio-UTP, [o~-32p]UTP or any other 32p-labeled ribonucleoside triphosphate can be used to synthesize the antisense or sense RNA probe. The 32p-labeled probes allow shorter exposure times and are less expensive to synthesize, but provide less autoradiographic resolution compared to the 35S-labeled probes. Although we generally use probes of high specific activity, the amount and specific activity of32p - or 35S-labeled ribonucleotide used to synthesize the RNA probe can be altered depending on how rapidly one wants to detect a hybridization signal and how abundant the mRNA is within the tissue section.
Hybridization o f R N A to Tissue Print Prior to hybridization, dried tissue prints should be washed for 4-12 hr in 0.1-0.2 x SSC containing 1% (w/v) SDS at 65°. After this washing step, prehybridization and hybridization are carried out in 1.5 x SSPE, 1% (w/v) SDS, 1% (w/v) nonfat powdered milk, 0.5 mg/ml denatured, sonicated salmon DNA, and 100 mM dithiothreitol at 68 °. We generally carry out prehybridization of the tissue print membranes for 12-16 hr, and follow this by hybridization with the antisense or sense RNA probes at 5 × 10 7 counts per minute (cpm)/ml in fresh buffer for 12-24 hr. After hybridization, tissue prints should be rinsed briefly in 2 x SSC, 1% SDS, and 10 mM dithiothreitol, and then washed two more times in fresh changes of 2 × SSC, I% SDS, and 10 mM dithiothreitol for 30 min each at 42 ° with gentle shaking. Two additional washes should be carried
[49]
TISSUE-PRINT HYBRIDIZATION FOR DETECTING R N A
693
out in 0.2 x SSC, 1% SDS, and 1 mM dithiothreitol at 65° for 30 min each. The membrane can then be dried and analyzed by autoradiography. As an alternative to the prehybridization and hybridization buffer described above, we have substituted a buffer containing 50% (v/v) formamide, 5 x Denhardt's solution, 8 6x SSC, 2 mM EDTA, 0.1% SDS, 200/.~g/ml poly(A), 100/~g/ml yeast tRNA, and 70 mM dithiothreitol. With this buffer system, prehybridization and hybridization should be carried out at 42° . Filters should be washed as described above following hybridization.
Staining Procedure Tissue prints can be stained with India ink2 or other dyes before autoradiography. Before staining, tissue prints should be briefly rinsed in icecold water and then immersed in ice-cold India ink (Higgins No. 4415; Faber-Castell Corporation) for 1-10 rain. Tissue prints can be destained by briefly rinsing in ice water and then by rinsing several times in 0.2 x SSC and 1% SDS. Although we have found that the tissue prints are not always uniformly stained with India ink, the stained images, nevertheless, provide a useful comparison to the autoradiographic images. The ink-stained images reveal anatomical detail that is not obvious in the autoradiograms, and provide an image for better interpretation of localized mRNA expression patterns on the autoradiograms. The ink-stained images are also useful for interpreting autoradiograms in terms of ineffective, incomplete, or distorted blotting of the sections onto the membrane.
Autoradiography Procedure We use Kodak XRP-5 film exposed for 24-48 hr at - 7 0 ° to evaluate initially the quality and quantity of hybridization to the tissue prints. Although exposure of the prints on XRP-5 is suitable for some tissue prints, autoradiograms of higher resolution can be obtained by exposing the tissue prints to the photographic film, Tmax 400. Exposure times on Tmax 400 are about five times longer than tissue prints exposed on XRP-5, but a higher quality image is obtained with the Tmax 400. With SAUR (small auxin up-regulated RNA) 4probes shown in Fig. 1, the autoradiograms on Tmax 400 were exposed for about 10 days at - 7 0 °. We develop the film for 6-11 min at 24° in Kodak Tmax developer. This is followed by Kodak stop bath for 30 sec and Kodak rapid fix for 5 min. The film is then washed in running tap water for 5 min, rinsed in Kodak, Photo-Flo 200, and air dried.
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OTHER METHODS
[49]
FIG. 1. Tissue-print hybridization reveals gravity-responsive mRNAs in soybean seedlings. Soybean seedlings were grown in the normal, vertical orientation or placed in a horizontal orientation for 20 rain prior to tissue planting. The reorientation of the soybean seedlings from the vertical to the horizontal position results in negative gravitropic curvature 20-45 min after the seedlings are placed in the horizontal position. Auxin-responsivernRNAs are detected in the epidermis and cortex tissues of the elongating region of the vertical hypocotyl with an SAUR antisense RNA hybridization probe labeled with [35S]thio-UTP? Tissue-print hybridization shows that the SAUR mRNAs disappear from the top of the horizontal hypocotyl prior to gravitropic bending. Schematic representations of the soybean seedlings are shown to the left or above the autoradiograms.
We have tested a number of other films for exposures of tissue-print hybridizations. K o d a k Tri-X Pan does not perform as well as Tmax 400, and exposure time for K o d a k Tmax 3200 is not substantially faster than Tmax 400. We have obtained the highest quality images with K o d a k Tech Pan 2415 (35-mm format) or K o d a k Tech Pan 4415 (4 × 5 in. format). The film speeds o f Tech Pan 2415 and 4415 film are considerably slower than Tmax 400. Tech Pan films have a fine grain and produce high-quality autoradiograms of tissue prints, but the slow speed of these films limits their application to tissue prints that have strong hybridization signals.
Comments Tissue-print hybridization provides an alternative method to in situ hybridization with fixed tissue sections. Tissue-print hybridization provides a reliable method to detect organ-specific and tissue-specific gene
[50]
CLONING FROM DRIED AGAROSE GELS
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expression because the patterns of hybridization observed are similar, if not identical, to those observed with in situ hybridization. 3'5'6'1° Tissueprint hybridization is much less time consuming and less expensive than in situ hybridization, and requires a minimal amount of technical expertise and equipment. Tissue-print hybridization also has the advantage that numerous tissue treatments or manipulations can be examined with a minimal amount of effort. For example, all of the tissue sections can be printed on a single piece of nylon membrane and, once the tissue prints have been made for each treatment or manipulation, staining, prehybridization, hybridization, and autoradiography can be uniformly carried out on that single nylon membrane. 10 M. A. Gee, G. Hagen, and T. J. Guilfoyle, Plant Cell 3, 419 (1991),
[50] R e c o v e r y a n d C l o n i n g o f G e n o m i c D N A F r a g m e n t s from Dried Agarose Gels By
MICHAEL W. MATHER,
J.
ANDREW KEIGHTLEY,
and JAMES A.
FEE
We describe here a method for the cloning of bacterial genes that circumvents the need to prepare and maintain genomic DNA libraries. Genomic DNA samples, highly enriched in specific restriction fragments, are isolated from dried agarose gels and used directly for cloning. 1 The method is rapid and technically simple, and reduces the possibility that a DNA sequence of interest might be missed due to underrepresentation in a library. Principle of the Method An analytical in-gel hybridization is employed to detect those genomic DNA restriction fragments that contain the sequence(s) of interest. An enriched fraction is subsequently isolated from a preparative-scale dried agarose gel, ligated into an appropriate vector, and propagated in Escherichia coli, Escherichia coil clones containing the fragment of interest are identified by a low-density colony hybridization screen. Hybridization in dried agarose gels was first described by Shinnick et al. a The technique is rapid, versatile, and sensitive. With radiolabeled l M. W. Mather, BioTechniques 6, 444 (1988). 2 T. M. Shinnick, E. Lurid, O. Smithies, and F. R. Blattner, Nucleic Acids Res. 2, 1911 (1975).
METHODS IN ENZYMOLOGY,VOL. 218
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