REVIEW |
Correspondence to: Ernst J.M. Speel, Dept. of Pathology, U. of Zürich, Schmelzbergstr. 12, CH-8091 Zürich, Switzerland.
![]() |
Summary |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In situ hybridization (ISH) has proved to be an invaluable molecular tool in research and diagnosis to visualize nucleic acids in their cellular environment. However, its applicability can be limited by its restricted detection sensitivity. During the past 10 years, several strategies have been developed to improve the threshold levels of nucleic acid detection in situ by amplification of either target nucleic acid sequences before ISH (e.g., in situ PCR) or the detection signals after the hybridization procedures. Here we outline the principles of tyramide signal amplification using the catalyzed reporter deposition (CARD) technique, present practical suggestions to efficiently enhance the sensitivity of ISH with CARD, and discuss some applications and possible future directions of in situ nucleic acid detection using such an amplification strategy. (J Histochem Cytochem 47:281288, 1999)
Key Words: (fluorescence) in situ hybridization, tyramide, signal amplification, mRNA, DNA, catalyzed reporter deposition, horseradish peroxidase, interphase cytogenetics, enzyme cytochemistry
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In situ hybridization (ISH) has proved to be an invaluable molecular tool in research and diagnosis and has significantly advanced the study of gene structure and expression at the level of individual cells in cell preparations as well as complex structural tissues (
In recent years, therefore, several strategies have been developed to amplify the nucleic acid targets (target amplification) or (immuno)cytochemical detection signals (signal amplification) in situ (Table 1). In addition, increased absolute amounts of hybridized probes were used successfully to improve the ISH detection sensitivity, e.g., cocktails of oligonucleotides or multiple cRNA probes (
|
![]() |
Target Amplification |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In general, target amplification techniques combine PCR and ISH to visualize specific amplified DNA and RNA sequences in cell and tissue preparations. In theory, these in situ PCR techniques are straightforward and include sample fixation and pretreatment that allows the primers, nucleotides, and polymerase enzymes to enter the cell but avoids the loss of the generated amplificants, PCR amplification in the cell, and direct or indirect (by ISH) detection of the amplificants. The practical procedure, however, is associated with several obstacles, such as low amplification efficiency (restricted sensitivity), poor reproducibility (restricted specificity), and difficulties in quantification of the results (
In situ PCR must therefore still be considered a rather cumbersome ISH method, in which sample pretreatment consists of fixation and protease digestion in combination with heating (thermal cycling) during nucleic acid amplification (by PCR). Moreover, the increase in detection sensitivity compared with conventional ISH is rather limited, even after optimization of the procedures (
In summary, initial enthusiasm has now changed into a more realistic and sometimes skeptical attitude about the practical potential of in situ PCR in research and clinical studies. As a consequence, in situ signal amplification approaches have been more and more explored during the past couple of years (Table 1), of which the CARD signal amplification method appears to be the most promising.
![]() |
CARD Signal Amplification |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
CARD signal amplification was introduced by
|
![]() |
Applications of In Situ CARD Signal Amplification |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
CARD signal amplification with biotinylated tyramides has been easily adapted for immunohistochemistry by
Since 1995, CARD has further been implemented in detection procedures for both DNA and RNA ISH on cell preparations and tissue sections. With signal amplification, the ISH sensitivity could be improved in the range of two- to 100-fold, enabling the detection of (a) repetitive and single-copy (up to the level of 15 KB) DNA sequences in cell preparations, (b) up to three different DNA sequences (repetitive as well as single-copy) simultaneously in cell preparations (
|
![]() |
Practical Suggestions for ISH Using CARD Signal Amplification |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The great advantage of using CARD signal amplification in ISH procedures is that it is applied after probe hybridization and stringent washings, so that the specificity of the probe hybridization is warranted. Therefore, in principle, proper conditions for sample fixation and pretreatment (including endogenous peroxidase inactivation) as well as hybridization in situ do not need to be modified compared with conventional ISH procedures. Only if nonspecific probe binding to the samples is suspected on the basis of control experiments should the probe concentration be further modified (decreased) for optimal results, e.g., in cases where complex DNA probes are used (e.g., chromosome painting, YAC, P1 or cosmid probes containing repetitive elements that need to be blocked by competitor DNA, such as Cot-I DNA) and an optimal balance is needed between probe and competitor DNA (
With respect to cytochemical probe detection, closer examination of the literature as well as Table 2 shows that a number of different combinations of probe detection (one to three detection conjugate layers) and CARD signal amplification systems (utilizing different tyramides, amplification buffers, reaction times, and temperatures) were applied, and that optimization of each detection system was necessary to obtain a high signal-to-noise ratio. Because with CARD signal amplification both specific and nonspecific (background) ISH signals are greatly amplified, it is essential for nonspecific probe binding (see above) and detection to be avoided or kept at a minimum to apply this procedure successfully. Therefore, we recommend routine optimization of probe hybridization (if not already done for setting up conventional ISH), cytochemical probe detection, and CARD signal amplification for one's own experiment. For this purpose, an appropriate one- to three-step detection system should be selected and combined with either a commercially available signal amplification kit (available from NEN Life Science Products (Boston, MA) as Tyramide Signal Amplification (TSA) kits or from Dako (Glostrup, Denmark) as Genpoint kit used according to the manufacturers' instructions, or CARD signal amplification using hapten- or fluorochrome-labeled tyramides diluted in an appropriate buffer, such as PBS buffer at pH 7.6 containing 0.1 M imidazole and 0.001% H2O2 (
|
We have recently introduced CARD signal amplification for our diagnostic nonradioactive oligonucleotide ISH procedure with digoxigenin-labeled probes to increase the sensitivity of the assay and to shorten the overall turnaround time of the assay (
Although the increase in ISH sensitivity by using CARD signal amplification is obvious from the literature, speculation about the obtained amplification factor is difficult, because thus for only very few quantitative evaluations have been carried out. Moreover, because the tyramide deposition reaction runs very quickly, minor differences in amplification reaction time may lead to variations in the final signal intensities. Nevertheless, an amplification factor in the range of five- to tenfold, or possibly higher, together with preservation of distinct localization of ISH signals, seems to be a realistic indication for both DNA and mRNA ISH.
In conclusion, CARD signal amplification using labeled tyramides has significantly influenced the ISH methods performed on different biological specimens, because it is an easy, rapid, highly sensitive, and efficient procedure. As a consequence, sensitive ISH procedures incorporating CARD signal amplification may become the tool of choice for low-copy nucleic acid and antigen detection in situ and therefore may be suitable for diagnostic laboratories. In addition, these procedures will facilitate the analysis of DNA ISH signals, e.g., in the evaluation of chromosomal aberrations in cytological and histological materials (visualization with low-magnification objectives), and might help advance the development of automated ISH spot-counting by computer-assisted image generation and analysis. We expect that the now available spectrum of probe labels, detection systems, and tyramide conjugates for CARD signal amplification will simplify and further improve ISH and will promote multiple-target nucleic acid detection in situ and procedures combining ISH and immunophenotyping.
![]() |
Acknowledgments |
---|
We thank P. Saremaslani for excellent technical assistence, N. Wey for computer-assisted reproductions, and Profs F.C.S Ramaekers (Department of Molecular Cell Biology and Genetics, University Maastricht, The Netherlands), Ph.U. Heitz, and J. Roth for continuous support.
Presented at the Histochemical Society Workshop, "Merging molecular biology with morphological techniques, in situ hybridization for the cellular localization of mRNA and DNA: State of the art 1998," July 23, 1998, in San Diego, CA.
Received for publication November 4, 1998; accepted November 24, 1998.
![]() |
Literature Cited |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Adams JC (1992) Biotin amplification of biotin and horseradish peroxidase signals in histochemical stains. J Histochem Cytochem 40:1457-1463
Adler K, Erickson T, Bobrow M (1997) High sensitivity detection of HPV-16 in SiHa and CaSki cells utilizing FISH enhanced by TSA. Histochem Cell Biol 108:321-324[Medline]
Bains MA, Agarwal R, Pringle JH, Hutchinson RM, Lauder I (1993) Flow cytometric quantitation of sequence-specific mRNA in hemopoietic cell suspensions by primer-induced in situ (PRINS) fluorescent nucleotide labeling. Exp Cell Res 208:321-326[Medline]
Berghorn KA, Bonnett JH, Hoffman GE (1994) cFos immunoreactivity is enhanced with biotin amplification. J Histochem Cytochem 42:1635-1642
Bobrow MN, Harris TD, Shaughnessy KJ, Litt GJ (1989) Catalyzed reporter deposition, a novel method of signal amplification. Application to immunoassays. J Immunol Methods 125:279-285[Medline]
Bobrow MN, Litt GJ, Shaughnessy KJ, Mayer PC, Conlon J (1992) The use of catalyzed reporter deposition as a means of signal amplification in a variety of formats. J Immunol Methods 150:145-149[Medline]
Bobrow MN, Shaughnessy KJ, Litt GJ (1991) Catalyzed reporter deposition, a novel method of signal amplification. J Immunol Methods 137:103-112[Medline]
Chao J, DeBiasio R, Zhu Z, Giuliano KA, Schmidt BF (1996) Immuno-fluorescence signal amplification by the enzyme-catalyzed deposition of a fluorescent reporter substrate (CARD). Cytometry 23:48-53[Medline]
Chen B-X, Szabolcs MJ, Matsushima AY, Erlanger BF (1996) A strategy for immunohistochemical signal enhancement by end-product amplification. J Histochem Cytochem 44:819-824
Chen RH, Fuggle SV (1993) In situ cDNA polymerase chain reaction. A novel technique for detecting mRNA expression. Am J Pathol 143:1527-1534[Abstract]
Chiu K-P, Cohen SH, Morris DW, Jordan GW (1992) Intracellular amplification of proviral DNA in tissue sections using the polymerase chain reaction. J Histochem Cytochem 40:333-341
De Haas RR, Verwoerd NP, Van der Corput MP, Van Gijlswijk RP, Siitari H, Tanke HJ (1996) The use of peroxidase-mediated deposition of biotintyramide in combination with time-resolved fluorescence imaging of europium chelate label in immunohistochemistry and in situ hybridization. J Histochem Cytochem 44:1091-1099
Dirks RW (1996) RNA molecules lighting up under the microscope. Histochem Cell Biol 106:151-166[Medline]
Femino AM, Fay FS, Fogarty K, Singer RH (1998) Visualization of single RNA transcripts in situ. Science 280:585-590
Forozan F, Karhu R, Kononen J, Kallioniemi A, Kallioniemi O-P (1997) Genome screening by comparative genomic hybridization. Trends Genet 13:405-409[Medline]
Gosden J, Hanratty D (1993) PCR in situ: a rapid alternative to in situ hybridization for mapping short, low copy number sequences without isotopes. Biotechniques 15:78-80[Medline]
Haase AT, Retzel EF, Staskus KA (1990) Amplification and detection of lentiviral DNA inside cells. Proc Natl Acad Sci USA 87:4971-4975[Abstract]
Hindkjaer J, Koch J, Terkelsen C, Brandt CA, Kolvraa S, Bolund L (1994) Fast, sensitive multicolor detection of nucleic acids in situ by primed in situ labeling (PRINS). Cytogenet Cell Genet 66:152-154[Medline]
Höfler H (1993) In situ polymerase chain reaction: toy or tool? Histochemistry 99:103-104[Medline]
Höfler H, Childers H, Montminy MR, Lechan RM, Goodmann RH, Wolfe HJ (1986) In situ hybridization methods for the detection of somatostatin mRNA in tissue sections using antisense RNA probes. Histochem J 18:597-604[Medline]
Höfler H, Pütz B, Mueller J, Neubert W, Sutter G, Gais P (1995) In situ amplification of measles virus RNA by the self-sustained sequence replication reaction. Lab Invest 73:577-585[Medline]
Hopman AHN, Kamps M, Dederen J, Speel EJM, Ramekers FCS (1998a) Bicolor ISH using CARD amplification for the rapid and efficient analysis of chromosomal imbalances in cancer. Cytometry Suppl 9:146-147
Hopman AHN, Ramaekers FCS, Speel EJM (1998b) Rapid synthesis of biotin-, digoxigenin-, trinitrophenyl-, and fluorochrome-labeled tyramides and their application for in situ hybridization using CARD-amplification. J Histochem Cytochem 46:771-777
Hopman AHN, Voorter CEM, Speel EJM, Ramaekers FCS (1997) In situ hybridization and comparative genomic hybridization. In Wolman SR, Sell S, eds. Cytogenetic Cancer Markers. Totowa, NJ, Humana Press, 45-69
Jacobs W, Dhaene K, Van Marck E (1998) Tyramine-amplified immunohistochemical testing using "homemade" biotinylated tyramine is highly sensitive and cost-effective. Arch Pathol Lab Med 122:642-643[Medline]
Kerstens HMJ, Poddighe PJ, Hanselaar AGJM (1995) A novel in situ hybridization signal amplification method based on the deposition of biotinylated tyramine. J Histochem Cytochem 43:347-352
Komminoth P (1996) Detection of mRNA in tissue sections using Dig-labeled RNA and oligonucleotide probes. In Non-radioactive In Situ Hybridization Application Manual. Mannheim, Boehringer Mannheim GmbH, pp. 126135
Komminoth P, Adams V, Long AA, Roth J, Saremaslani P, Flury R, Schmid M, Heitz PU (1994) Evaluation of methods for hepatitis C virus detection in archival liver biopsies. Comparison of histology, immunohistochemistry, in-situ hybridization, reverse transcriptase polymerase chain reaction (RT-PCR) and in-situ RT-PCR. Pathol Res Pract 190:1017-1025[Medline]
Komminoth P, Long AA (1993) In-situ polymerase chain reaction. An overview of methods, applications and limitations of a new molecular technique. Virchows Arch [B] 64:67-73[Medline]
Komminoth P, Werner M (1997) Target and signal amplification: approaches to increase the sensitivity of in situ hybridization. Histochem Cell Biol 108:325-333[Medline]
Lichter P (1997) Multicolor FISHing: what's the catch? Trends Genet 13:475-479[Medline]
Long AA, Komminoth P (1997) In situ PCR. In Gosden JR, ed. PRINS and In Situ PCR protocols. Methods in Molecular Biology. Vol 71. Totowa, NJ, Humana Press, 141161
Macechko PT, Krueger L, Hirsch B, Erlandsen SL (1997) Comparison of immunologic amplification vs enzymatic deposition of fluorochrome-conjugated tyramide as detection systems for FISH. J Histochem Cytochem 45:359-363
Mayer G, Bendayan M (1997) Biotinyltyramides: a novel approach for electron microscopic immunocytochemistry. J Histochem Cytochem 45:1449-1454
McKay JA, Murray GI, Keith WN, McLeod HL (1997) Amplification of fluorescent in situ hybridisation signals in formalin fixed paraffin wax embedded sections of colon tumour using biotinylated tyramide. J Clin Pathol Mol Pathol 50:322-325[Abstract]
McNicol AM, Farquharson MA (1997) In situ hybridization and its diagnostic applications in pathology. J Pathol 182:250-261[Medline]
Mee AP, Denton J, Hoyland JA, Davies M, Mawer EB (1997) Quantification of vitamin D receptor mRNA in tissue sections demonstrates the relative limitations of in situ reverse transcriptase polymerase chain reaction. J Pathol 182:22-28[Medline]
Merz H, Malisius R, Mannweiler S, Zhou R, Hartmann W, Orscheschek K, Moubayed P, Feller AC (1995) Immunomax. A maximized immunohistochemical method for the retrieval and enhancement of hidden antigens. Lab Invest 73:149-156[Medline]
Mogensen J, Kolvraa S, Hindkjær J, Petersen S, Koch J, Nygård M, Jensen T, Gregersen N, Junker S, Bolund L (1991) Nonradioactive, sequence-specific detection of RNA in situ by primed in situ labeling (PRINS). Exp Cell Res 196:92-98[Medline]
Nuovo GJ (1992) PCR In Situ Hybridization. Protocols and Applications. New York, Raven Press
Odinot PT, Meis JFGM, HoogkampKorstanje JAA, Melchers WJG (1998) In situ localisation of Yersinia enterocolitica by catalysed reporter deposition signal amplification. J Clin Pathol 51:444-449[Abstract]
O'Leary JJ, Chetty R, Graham AK, McGee J, O'D (1996) In situ PCR: pathologist's dream or nightmare? J Pathol 178:11-20[Medline]
Pastore JN, Clampett C, Miller J, Porter K, Miller D (1995) A rapid immunoenzyme double labeling technique using enzyme polymer system (EPOS) reagents. J Histotechnol 18:35-40
Pilling A, EndersbyWood H, Jones S (1997) Increased sensitivity of in situ hybridization: implications for in situ amplification. Diagn Mol Pathol 6:174[Medline]
Plenat F, Picard E, Antunes L, Vignaud J-M, Marie B, Chalabreysse P, Muhale F (1997) L'amplification des réactions immunologiques par dépot catalytique aux sites de réaction de dérivés de la tyramine. Ann Pathol 17:17-23[Medline]
Poddighe PJ, Bulten J, Kerstens HMJ, Robben JCM, Melchers WJG, Hanselaar AGJM (1996) Human papilloma virus detection by in situ hybridisation signal amplification based on biotinylated tyramine deposition. Clin Mol Pathol 49:M340-344
Raap AK, Van de Corput MPC, Vervenne RAW, Van Gijlswijk RPM, Tanke HJ, Wiegant J (1995) Ultra-sensitive FISH using peroxidase-mediated deposition of biotin- or fluorochrome tyramides. Hum Mol Genet 4:529-534[Abstract]
Reed JA, Nador RG, Spaulding D, Tani Y, Cesarman E, Knowles DM (1998) Demonstration of Kaposi's sarcoma-associated Herpes virus cyclin D homolog in cutaneous Kaposi's sarcoma by colorimetric in situ hybridzation using catalyzed signal amplification system. Blood 91:3825-3832
Sällström JF, Zehbe I, Alemi M, Wilander E (1993) Pitfalls of in situ polymerase chain reaction (PCR) using direct incorporation of labelled nucleotides. Anticancer Res 13:1153[Medline]
Sanno N, Teramoto A, Sugiyama M, Itoh Y, Osamura RY (1996) Application of catalyzed signal amplification in immunodetection of gonadotropin subunits in clinically nonfunctioning pituitary adenomas. Am J Clin Pathol 106:16-21[Medline]
Schmidt BF, Chao J, Zhu Z, DeBiasio RL, Fisher G (1997) Signal amplification in the detection of single-copy DNA and RNA by enzyme-catalyzed deposition (CARD) of the novel fluorescent reporter substrate Cy3.29tyramide. J Histochem Cytochem 45:365-373
Schöfer C, Weipoltshammer K, Almeder M, Wachtler F (1997) Signal amplification at the ultrastructural level using biotinylated tyramides and immunogold detection. Histochem Cell Biol 108:313-319[Medline]
Shindler KS, Roth KA (1996) Double immunofluorescent staining using two unconjugated primary antisera raised in the same species. J Histochem Cytochem 44:1331-1335
Speel EJM, Hopman AHN, Komminoth P (in press) Signal amplification for DNA and mRNA in situ hybridization. In Darby J, ed. In Situ Hybridization Protocols. Methods in Molecular Biology. Totowa, NJ, Humana Press
Speel EJM, Lawson D, Hopman AHN, Gosden J (1995a) Multi-PRINS: multiple sequential oligonucleotide primed in situ DNA synthesis reactions label specific chromosomes and produce bands. Hum Genet 95:29-33[Medline]
Speel EJM, Lawson D, Ramaekers FCS, Gosden JR, Hopman AHN (1996) Rapid brightfield detection of oligonucleotide primed in situ (PRINS)-labeled DNA in chromosome preparations and frozen tissue sections. Biotechniques 20:226-234[Medline]
Speel EJM, Ramaekers FCS, Hopman AHN (1995b) Detection systems for in situ hybridization, and the combination with immunocytochemistry. Who is still afraid of red, green and blue? Histochem J 27:833-858[Medline]
Speel EJM, Ramaekers FCS, Hopman AHN (1997) Sensitive multicolor fluorescence in situ hybridization using catalyzed reporter deposition (CARD) amplification. J Histochem Cytochem 45:1439-1446
Speel EJM, Saremaslani P, Roth J, Hopman AHN, Komminoth P (1998) Improved mRNA in situ hybridization on formaldehyde-fixed and paraffin-embedded tissue using signal amplification with different haptenized tyramides. Histochem Cell Biol 110:571-577[Medline]
Sperry A, Jin L, Lloyd RV (1996) Microwave treatment enhances detection of RNA and DNA by in situ hybridization. Diagn Mol Pathol 5:291-296[Medline]
Tecott LH, Barchas JD, Eberwine JH (1988) In situ transcription: specific synthesis of complementary DNA in fixed tissue sections. Science 240:1661-1664[Medline]
Teo IA, Shaunak S (1995) Polymerase chain reaction in situ: an appraisal of an emerging technique. Histochem J 27:647-659[Medline]
Terkelsen C, Koch J, Kolvraa S, Hindkjær J, Pedersen S, Bolund L (1993) Repeated primed in situ labeling: formation and labeling of specific DNA sequences in chromosomes and nuclei. Cytogenet Cell Genet 63:235-237[Medline]
Trembleau A, Bloom FE (1995) Enhanced sensitivity for light and electron microscopic in situ hybridization with multiple simultaneous nonradioactive oligodeoxynucleotide probes. J Histochem Cytochem 43:829-841
Troyer DL, Goad DW, Xie H, Rohrer GA, Alexander LJ, Beattle CW (1994) Use of direct in situ single-copy (DISC) PCR to physically map five porcine microsatellites. Cytogenet Cell Genet 67:199-204[Medline]
Van Gijlswijk RPM, Wiegant J, Raap AK, Tanke HJ (1996a) Improved localization of fluorescent tyramides for fluorescence in situ hybridization using dextran sulfate and polyvinyl alcohol. J Histochem Cytochem 44:389-392
Van Gijlswijk RPM, Wiegant J, Vervenne R, Lasan R, Tanke HJ, Raap AK (1996b) Horseradish peroxidase-labeled oligonucleotides and fluorescent tyramides for rapid detection of chromosome-specific repeat sequences. Cytogenet Cell Genet 75:258-262[Medline]
Van Gijlswijk RPM, Zijlmans HJMAA, Wiegant J, Bobrow MN, Erickson TJ, Adler KE, Tanke HJ, Raap AK (1997) Fluorochrome-labeled tyramides: use in immunocytochemistry and fluorescence in situ hybridization. J Histochem Cytochem 45:375-382
Van Heusden J, De Jong P, Ramaekers FCS, Bruwiere H, Borgers M, Smets G (1997) Fluorescein-labeled tyramide strongly enhances the detection of low bromodeoxyuridine incorporation levels. J Histochem Cytochem 45:315-319
Von Wasielewski R, Mengel M, Gignac S, Wilkens L, Werner M, Georgii A (1997) Tyramine amplification technique in routine immunohistochemistry. J Histochem Cytochem 45:1455-1459
Werner M, Von Wasielewski R, Komminoth P (1996) Antigen retrieval, signal amplification and intensification in immunohistochemistry. Histochem Cell Biol 105:253-260[Medline]
Zaitsu K, Ohkura Y (1980) New fluorogenic substrates for horseradish peroxidase: rapid and sensitive assays for hydrogen peroxide and the peroxidase. Anal Biochem 109:109-113[Medline]
Zehbe I, Hacker GW, Sällström JF, Rylander E, Wilander E (1994) Self-sustained sequence replication-based amplification (3SR) for the in situ detection of mRNA in cultured cells. Cell Vis 1:20-24
Zehbe I, Hacker GW, Su H, HauserKronberger C, Hainfeld JF, Tubbs R (1997) Sensitive in situ hybridization with catalyzed reporter deposition, streptavidin-nanogold, and silver acetate autometallography. Am J Pathol 150:1553-1561[Abstract]