- Email: [email protected]
A rapid method for FISH analysis on interphase nuclei extracted from cryofixed tissues
TECHNICAL
TIPS
FxatJtm1. Profile of protein tyrosine phosphatases (PTPs) expressed in adult mouse brain, lung and kidney as revealed by digestion of amplification products with Alul and R.ml. LMI and LAR-2refer to the mouse homologs of rat PTPs cloned by Zhang et al.4, while PTPID (Syp) refers to the mouse PTP cloned by Feng et al.5 RT-PCR was performed on total RNA according to conventional procedures using a 3ZP-endlabeled 5' primer (GGGAATrCAARTGYGMICARTAYrGGCC)and unlabeled 3' primer (CGGATCCAYICCIGCRCTRCARTG). PCR products of the expected size (300 bp) were purified following electrophoresis through 2% SeaKem LE agarose using the QIAEX kit (Qiagen). A tenth of each sample was then digested with Alul or RsaI. In a modification to Boehm's protocolt, samples were fractionated on a 6% sequencing gel rather than on a native gel. The improved resolution, coupled with the use of MI 3 sequencing tracks as ladders, made it possible, in many instances, to assign identities to bands based on published sequence data. The gel was dried without fixation before autoradiographicexposure for 2-4 h at room tempetamre.
0It
In all instances, sequencing of cloned fragments established that they were homologous to or identical to known PTPs. The identities of three of these PTPs are indicated in Fig. 1. The procedure has allowed cloning even of bands that :ave faint signals on displays, representing less than 1% of total specific RT-PCR ~roduct. In the original method t, eDNA fragments were eluted from the display gel, labeled by asymmetric PCR in the presence of radiolabeled deoxynucleotide and used to probe northern blots or to screen libraries. However, in our hands, these probes were usually of low specific activity and unsuitable for detection of lowabundance transcripts. In addition, what appears to be a unique band on the display gel may contain unlabeled digestion products of the same size or traces of unresolved fragments. Copurification of contaminating fragments may lead to difficulties in generating specific probes that will recapitulate the difference in transcript levels demonstrated in the display3. Both of these limitations can be overcome if bands of interest are cloned directly by the method described.
Ooa°'°
Alul
ACKNOWLEDGFM EN'IN This study was carried out in the laborato~, of Austin Smith, and supported by rite Biotechnologyand Biological Sciences Research Council of the United Kingdom. REFERENCF2; 1 Boehm, T. (1993) Oncogene8. 1385-1390 2 Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989)MolecularCloning: A Laborato~. Manual, Cold Spring Harbor Laboratory Press 3 Li, F., Bamathan, E.S. attd Kariko, K. (1994) Nacleic Acids Res. 22, 1764-1765 4 Zhang, w.g. et al. (1994) BiochemJ. 302, 39-47 5 Feng, G.S., ltui, C.C. and Pawson, T. (199.3) Science 259, 1607-1611
Rsal
o o •
o°O
Comributed by Kenneth Lee ([email protected]), Centre for Genome Research, King's Buildings, University of Edinburgh, Edinburgh, OK EH9 3JQ.
A rapid method for FISH analysis o n interphase nuclei extracted from cryoflxed tissues The release of nuclei from archived tissue was first described by Hedley et al. in 1983 (Ref. 1). However, this protocol, which has subsequently been modified by various authors (e.g. Refs 2-4), was for paraffin-embedded material only. We describe a method to extract well conserved nuclei, suitable for fluorescence in sittt hydridization (FISH) analysis, from cryoflxed material stored at -80°C for several yeats. Use of this material avoids time-consuming and laborious dewaxing and rehydtation procedures needed when handling paraffin-embedded tissue, cutting working time by a third. The main modification compared with the standard protocol is the initial fixation of the cryosections with pata formaldehyde. The protocol is then the same as described for paralTm-embedded tissue (Ref. 4), starting with proteinase K treatment. The resulting nuclei are suitable for two- or three-colour FISH with centromere (Fig. la) as well as cosmid probes down to 10 kb in size (Fig. l b). The efficier, cy of extracting 71o~.ieiwas as bigh as reported for pe.ra.W:nembedded tissue"-; 15 25 ~.m sections of cryofixed tissue (2-5 mm in diameter), provide sufficient material for 5-10 slides. We were able to extract nuclei of all 10 tested cases. Two types of tissues have been used: sural-nerve biopsies (10 of the 10 cases) and muscle biopsies (2 of the 10 cases), which have been archived for between I and 6 yeats at -80°C. The efficiency of nucleus extraction and suitability for FISH was independent of the tissue type. Probes used for FISH have either been obtained digoxigenated or biotinylated from Oncor (centromere probes) or were labelled with their ligands by nick translation (cosmids). Three-colour FISH has been performed for the cemt omere probes, only two colour~ have been used for the cosmids. The percentage of nuclei that coutd not be evaluated because of background or the lack of one or more of the expected signals per nucleus was slighdy higher for the centromere probes (10-20%) [for details TIG DECEMBER 1996 VOL, 12 NO. 12
505
TECHNICAL
TIPS
see Re['. 5; Table 2], while the cosmid probes were within the expected range (10-50% Refs 4, 6). Less than 4°~ of the nuclei considered for evaluation showed more than the expected four or six signals. Fifty nuclei per case have been evaluated by fluorescence microscope. Protocol 1 About 15 25 p.m cryosections (2-5 mm in diameter) are collected in a 1.5 ml tube (cooled down to approximately -30°C in the cryostat) and immediately afterwards fixed in 1 ml fresh paraformaldehyde (PFA) solution [4% in phosphate-buffered saline (PBS; Biochrom, L182-10)} for 2-3 h at room temperature. The tissue should thaw as soon as the PFA solution 1. Typical nuclei released out of cryois added. fixed sural-nerve biopsy tissue of a healthy 2 After fixation, the samples are washed twice with 0.9o/0NaCI (1 ml) for a few female donor after the FISH procedure. The seconds at room temperature. Sedimentation of the tissue between these blue colour of the nuclei is the result of DAFt steps is done by centrifugation (1000 rev min-1, 3 min). counterstaining. Pictures are taken on a Zeiss 3 Mincing of the tissue, proteinase K treatment, removing the damaged cells Axiophot microscope equipped with an Imac from the nuclei, spotting on the slides and modifications for the FISH proCCD $30 camera (Compulog) using the softcedure are performed as described in Re['. 4. A more detailed standard FISH ware of Metasystems GmbH (Hardtstr. 51/1, protocol is described in Ref. 6. D-69207 Sandhausen, Germany) according to 4 For three-colour FISH, biotinylated and digoxigenated probes from Oncor manufacturers instmctions. (a) A nucleus after can be used. The first ones are detected by an avidin-fluoresceinisothiocyanate three-colour FISHhas been performed with the (avidin-FITC) system and lead to green signals, and the second ones by centromeric probes for the chromosomes 7 antidigoxigenln-rhodamine, producing red signals. By mixing biotinylated (green spots) [Oncorcatalogue no. P5OI1-B.5L and digoxigenated probes spedfic for the same chromosome in a 1:1 ratio, 8 (red spots) [P5013-DG.5] and 17 (yellow the third colour (yellow signal) can be produced (Fig. la). spots) [PSO40-B.5and PS040-DG.5L(b) A nucleus probed with the cosmid probes pVAW409R1 (10 kb; Ref. 7), located on 17plt.2 (green ACKNOX~ZEDGF.MENTS This work was funded by grants to B.R. and H.G. from the Deutsche spots) and cP,CNeu I (30 kb; Ref. 8), located on Forschungsgemeinschaft.T.L. holds a fellowship of the Herbert Quandt sriftung 17q11.12 (red spots). der Varta AG. We thank R. Cawthon (Salt Lake City, USA) for providing the probe cRCNeul and E. Gebhart, R.A. Pfeiffer, G. Fey and B. Neund/~rfer (Eflangen, Germany) for their continuous support. [I~FFERENCES
1 2 3 4 5 6 7 8
Hedley, D.W. etal. (1983).L Histochem. Cytochem. 31, 1333-1335 Amoldus, E.P.J. et al. (1991)J. Clin. Pathol. 44, 900-904 Lee, W. et al. (1993) Cancer Genet. Cytogenet. 68, 99-103 Liehr, T. el al. (1995) Trends Genet. 11, 377-378 Gebhart, E. et al. (1993) AnticancerRes. 13, 1857-1862 Liehr, T. etaL(1995) Appl. Cytogenet. 21,185-188 Barker. D. el al. (1978) Science 236, 1100-1102 Kallionlemi, O-P. et al. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 5321-5325
Contributed by Thomas Ltehr', Holger GrehP and Bernd Rautenstrauss* ([email protected]), *Institute for Hnman Genetics and ~Department of Neurologxv Schwabachanlage 10, 19-91054 Erlangen, Germany.
An improved protocol for purification of 35S-labelledoligonucleotide DNA probes for in stu~ h y b r i d i z a t i o n a p p l i c a t i o n s In situ hybridization analysis with oligonucleotide (oligo) DNA probes provides a flexible and powerful approach to analyse the spatial distribution of active genesk However, problems with background and low sensitivity have limited its wide application. We have compar~.xl the performance of labelled oligo DNA probes purified by a variety of methods. Our results show that 55Slabelled, p~ly(dA)-tailed oligo DNA probes purified by oligo(dT)-resin provide the highest sensitivity and also the lowest background. Figure 1 shows IGF2 expression in an 8-week-old human embryo analysed by an IGF2-specific oligo DNA probe. Oligo DNA probes purified by tile Mermaid kit (Bio 101, Inc.), for example, produced only a weak hybridization signal and high background (Fig. ld), whereas the oligo(dT)-resin-purified oligo DNA probe produced a much higher signal with less background (Fig. lc). The high fidelity of this strong hybridization signal was verified by comparing it with the hybridization signal obtained by using an antisense, 35S-labelled IGF2 RNA probe (Fig. lb) (Ref. 2). The high hybridization signal and low background of the oligo(dT)-resin-purified IGF2 oligo DNA probe is due not only to the efficient removal of proteins and radioactive nucleotide precursors, but also to the selection against unlabelled or shorttailed oligo DNAs. Figure 2 shows that the average size, and hence the specific radioactivity of the oligo probe, is dramatically improved hy the oligo(dT)-resin purification step. In addition to radioactive poly(dA)-tailed oligo probes, this method should also work with other, non-radioactive oligo probes with homopolymeric 3" ends. Protocol I Pretreat oligo(dT)-ceflulose Type 7 (Pharmacia) by suspending 0.1 g oligo(dT)-cellulose in 500 ~l distilled water, washing the resin with the same volume of 0.1 ~,tNaOH, five to eight times and equilibrating with 1x binding buffer (10 mMTris-HCI, "FIG DECEMBER 1996 VOL. 12 NO. 12
506
TIPS
FxatJtm1. Profile of protein tyrosine phosphatases (PTPs) expressed in adult mouse brain, lung and kidney as revealed by digestion of amplification products with Alul and R.ml. LMI and LAR-2refer to the mouse homologs of rat PTPs cloned by Zhang et al.4, while PTPID (Syp) refers to the mouse PTP cloned by Feng et al.5 RT-PCR was performed on total RNA according to conventional procedures using a 3ZP-endlabeled 5' primer (GGGAATrCAARTGYGMICARTAYrGGCC)and unlabeled 3' primer (CGGATCCAYICCIGCRCTRCARTG). PCR products of the expected size (300 bp) were purified following electrophoresis through 2% SeaKem LE agarose using the QIAEX kit (Qiagen). A tenth of each sample was then digested with Alul or RsaI. In a modification to Boehm's protocolt, samples were fractionated on a 6% sequencing gel rather than on a native gel. The improved resolution, coupled with the use of MI 3 sequencing tracks as ladders, made it possible, in many instances, to assign identities to bands based on published sequence data. The gel was dried without fixation before autoradiographicexposure for 2-4 h at room tempetamre.
0It
In all instances, sequencing of cloned fragments established that they were homologous to or identical to known PTPs. The identities of three of these PTPs are indicated in Fig. 1. The procedure has allowed cloning even of bands that :ave faint signals on displays, representing less than 1% of total specific RT-PCR ~roduct. In the original method t, eDNA fragments were eluted from the display gel, labeled by asymmetric PCR in the presence of radiolabeled deoxynucleotide and used to probe northern blots or to screen libraries. However, in our hands, these probes were usually of low specific activity and unsuitable for detection of lowabundance transcripts. In addition, what appears to be a unique band on the display gel may contain unlabeled digestion products of the same size or traces of unresolved fragments. Copurification of contaminating fragments may lead to difficulties in generating specific probes that will recapitulate the difference in transcript levels demonstrated in the display3. Both of these limitations can be overcome if bands of interest are cloned directly by the method described.
Ooa°'°
Alul
ACKNOWLEDGFM EN'IN This study was carried out in the laborato~, of Austin Smith, and supported by rite Biotechnologyand Biological Sciences Research Council of the United Kingdom. REFERENCF2; 1 Boehm, T. (1993) Oncogene8. 1385-1390 2 Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989)MolecularCloning: A Laborato~. Manual, Cold Spring Harbor Laboratory Press 3 Li, F., Bamathan, E.S. attd Kariko, K. (1994) Nacleic Acids Res. 22, 1764-1765 4 Zhang, w.g. et al. (1994) BiochemJ. 302, 39-47 5 Feng, G.S., ltui, C.C. and Pawson, T. (199.3) Science 259, 1607-1611
Rsal
o o •
o°O
Comributed by Kenneth Lee ([email protected]), Centre for Genome Research, King's Buildings, University of Edinburgh, Edinburgh, OK EH9 3JQ.
A rapid method for FISH analysis o n interphase nuclei extracted from cryoflxed tissues The release of nuclei from archived tissue was first described by Hedley et al. in 1983 (Ref. 1). However, this protocol, which has subsequently been modified by various authors (e.g. Refs 2-4), was for paraffin-embedded material only. We describe a method to extract well conserved nuclei, suitable for fluorescence in sittt hydridization (FISH) analysis, from cryoflxed material stored at -80°C for several yeats. Use of this material avoids time-consuming and laborious dewaxing and rehydtation procedures needed when handling paraffin-embedded tissue, cutting working time by a third. The main modification compared with the standard protocol is the initial fixation of the cryosections with pata formaldehyde. The protocol is then the same as described for paralTm-embedded tissue (Ref. 4), starting with proteinase K treatment. The resulting nuclei are suitable for two- or three-colour FISH with centromere (Fig. la) as well as cosmid probes down to 10 kb in size (Fig. l b). The efficier, cy of extracting 71o~.ieiwas as bigh as reported for pe.ra.W:nembedded tissue"-; 15 25 ~.m sections of cryofixed tissue (2-5 mm in diameter), provide sufficient material for 5-10 slides. We were able to extract nuclei of all 10 tested cases. Two types of tissues have been used: sural-nerve biopsies (10 of the 10 cases) and muscle biopsies (2 of the 10 cases), which have been archived for between I and 6 yeats at -80°C. The efficiency of nucleus extraction and suitability for FISH was independent of the tissue type. Probes used for FISH have either been obtained digoxigenated or biotinylated from Oncor (centromere probes) or were labelled with their ligands by nick translation (cosmids). Three-colour FISH has been performed for the cemt omere probes, only two colour~ have been used for the cosmids. The percentage of nuclei that coutd not be evaluated because of background or the lack of one or more of the expected signals per nucleus was slighdy higher for the centromere probes (10-20%) [for details TIG DECEMBER 1996 VOL, 12 NO. 12
505
TECHNICAL
TIPS
see Re['. 5; Table 2], while the cosmid probes were within the expected range (10-50% Refs 4, 6). Less than 4°~ of the nuclei considered for evaluation showed more than the expected four or six signals. Fifty nuclei per case have been evaluated by fluorescence microscope. Protocol 1 About 15 25 p.m cryosections (2-5 mm in diameter) are collected in a 1.5 ml tube (cooled down to approximately -30°C in the cryostat) and immediately afterwards fixed in 1 ml fresh paraformaldehyde (PFA) solution [4% in phosphate-buffered saline (PBS; Biochrom, L182-10)} for 2-3 h at room temperature. The tissue should thaw as soon as the PFA solution 1. Typical nuclei released out of cryois added. fixed sural-nerve biopsy tissue of a healthy 2 After fixation, the samples are washed twice with 0.9o/0NaCI (1 ml) for a few female donor after the FISH procedure. The seconds at room temperature. Sedimentation of the tissue between these blue colour of the nuclei is the result of DAFt steps is done by centrifugation (1000 rev min-1, 3 min). counterstaining. Pictures are taken on a Zeiss 3 Mincing of the tissue, proteinase K treatment, removing the damaged cells Axiophot microscope equipped with an Imac from the nuclei, spotting on the slides and modifications for the FISH proCCD $30 camera (Compulog) using the softcedure are performed as described in Re['. 4. A more detailed standard FISH ware of Metasystems GmbH (Hardtstr. 51/1, protocol is described in Ref. 6. D-69207 Sandhausen, Germany) according to 4 For three-colour FISH, biotinylated and digoxigenated probes from Oncor manufacturers instmctions. (a) A nucleus after can be used. The first ones are detected by an avidin-fluoresceinisothiocyanate three-colour FISHhas been performed with the (avidin-FITC) system and lead to green signals, and the second ones by centromeric probes for the chromosomes 7 antidigoxigenln-rhodamine, producing red signals. By mixing biotinylated (green spots) [Oncorcatalogue no. P5OI1-B.5L and digoxigenated probes spedfic for the same chromosome in a 1:1 ratio, 8 (red spots) [P5013-DG.5] and 17 (yellow the third colour (yellow signal) can be produced (Fig. la). spots) [PSO40-B.5and PS040-DG.5L(b) A nucleus probed with the cosmid probes pVAW409R1 (10 kb; Ref. 7), located on 17plt.2 (green ACKNOX~ZEDGF.MENTS This work was funded by grants to B.R. and H.G. from the Deutsche spots) and cP,CNeu I (30 kb; Ref. 8), located on Forschungsgemeinschaft.T.L. holds a fellowship of the Herbert Quandt sriftung 17q11.12 (red spots). der Varta AG. We thank R. Cawthon (Salt Lake City, USA) for providing the probe cRCNeul and E. Gebhart, R.A. Pfeiffer, G. Fey and B. Neund/~rfer (Eflangen, Germany) for their continuous support. [I~FFERENCES
1 2 3 4 5 6 7 8
Hedley, D.W. etal. (1983).L Histochem. Cytochem. 31, 1333-1335 Amoldus, E.P.J. et al. (1991)J. Clin. Pathol. 44, 900-904 Lee, W. et al. (1993) Cancer Genet. Cytogenet. 68, 99-103 Liehr, T. el al. (1995) Trends Genet. 11, 377-378 Gebhart, E. et al. (1993) AnticancerRes. 13, 1857-1862 Liehr, T. etaL(1995) Appl. Cytogenet. 21,185-188 Barker. D. el al. (1978) Science 236, 1100-1102 Kallionlemi, O-P. et al. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 5321-5325
Contributed by Thomas Ltehr', Holger GrehP and Bernd Rautenstrauss* ([email protected]), *Institute for Hnman Genetics and ~Department of Neurologxv Schwabachanlage 10, 19-91054 Erlangen, Germany.
An improved protocol for purification of 35S-labelledoligonucleotide DNA probes for in stu~ h y b r i d i z a t i o n a p p l i c a t i o n s In situ hybridization analysis with oligonucleotide (oligo) DNA probes provides a flexible and powerful approach to analyse the spatial distribution of active genesk However, problems with background and low sensitivity have limited its wide application. We have compar~.xl the performance of labelled oligo DNA probes purified by a variety of methods. Our results show that 55Slabelled, p~ly(dA)-tailed oligo DNA probes purified by oligo(dT)-resin provide the highest sensitivity and also the lowest background. Figure 1 shows IGF2 expression in an 8-week-old human embryo analysed by an IGF2-specific oligo DNA probe. Oligo DNA probes purified by tile Mermaid kit (Bio 101, Inc.), for example, produced only a weak hybridization signal and high background (Fig. ld), whereas the oligo(dT)-resin-purified oligo DNA probe produced a much higher signal with less background (Fig. lc). The high fidelity of this strong hybridization signal was verified by comparing it with the hybridization signal obtained by using an antisense, 35S-labelled IGF2 RNA probe (Fig. lb) (Ref. 2). The high hybridization signal and low background of the oligo(dT)-resin-purified IGF2 oligo DNA probe is due not only to the efficient removal of proteins and radioactive nucleotide precursors, but also to the selection against unlabelled or shorttailed oligo DNAs. Figure 2 shows that the average size, and hence the specific radioactivity of the oligo probe, is dramatically improved hy the oligo(dT)-resin purification step. In addition to radioactive poly(dA)-tailed oligo probes, this method should also work with other, non-radioactive oligo probes with homopolymeric 3" ends. Protocol I Pretreat oligo(dT)-ceflulose Type 7 (Pharmacia) by suspending 0.1 g oligo(dT)-cellulose in 500 ~l distilled water, washing the resin with the same volume of 0.1 ~,tNaOH, five to eight times and equilibrating with 1x binding buffer (10 mMTris-HCI, "FIG DECEMBER 1996 VOL. 12 NO. 12
506