Plant Molecular Biology Reporter 18: 65a–65f, 2000 2000 International Society for Plant Molecular Biology. Printed in Canada.
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Nonradioactive Identification of Differential Display RT-PCR Products MARIBEL COLMENARES*,1, CARLOS GIMÉNEZ2, MAIRA OROPEZA2 and EVA DE GARCÍA2 1 Laboratorio de Fisiología Vegetal, Departamento de Biología. Facultad Experimental de Ciencias. La Universidad del Zulia. Apartado 10488 Bella Vista, Maracaibo 4002, Venezuela; 2Laboratorio de Biotecnología Vegetal, Centro de Botánica Tropical, Instituto de Biología Experimental, Facultad de Ciencias, Universidad Central de Venezuela. Apartado 47114 Los Chaguaramos, Caracas 1040 A, Venezuela
Abstract. A simple, sensitive protocol for nonradioactive screening of DDRT-PCR candidates is described. Embryogenic and nonembryogenic cDNAs were prepared from mRNA isolated using paramagnetic particles. Reverse transcription was performed with biotinylated oligo-dT(18). These labeled cDNAs were checked for integrity and used as a probe for screening DDRT-PCR candidates. Most of the DDRT-PCR candidates detected were from mRNA expressed at low levels, giving weak or undetectable signals, and stronger signals represented more highly expressed mRNAs. Our results demonstrate the usefulness of chemiluminescence detection to screen DDRT-PCR products. This strategy is an alternative method to radioactive detection procedures. Key words: differential display, nonradioactive reverse northern, Saccharum sp., somatic embryogenesis Colmenares et al. Nonradioactive DDRT-PCR products
Introduction Differential display reverse transcription (DDRT) is a powerful procedure for detecting differentially expressed genes (Liang and Pardee, 1992; Liang et al., 1993). However, a high proportion of putative differential display (DD) candidates proves to be false positives. Analysis of DDRT-PCR gene expression requires time consuming screening of hundreds of candidates and limiting amounts of RNA often preclude analysis of all the candidates. Radioactive detection methods, commonly used to identify band patterns in differential display experiments, have the disadvantages of requiring handling of hazardous radioisotopes, potential contamination of the working area and thermal cycler, as well as the cost burden of radioactive waste disposal. As an alternative, Lohman et al. (1995) used polyacrylamide gels and visualized DNA bands by silver staining. The sensitivity of silver staining, however, is generally much lower than that obtained using radioisotopes. On the other hand, a chemiluminiscence-based differential display *Author for correspondence. e-mail:
[email protected]; fax: (5861)-515390.
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protocol has been described using sequencing gels and expression patterns were confirmed by northern blots analysis (An et al. 1996, Luehersen et al., 1997). Other modifications have been reported such as DDRT-PCR in agarose gels (Rompf and Kahl, 1997). Zegzouti et al. (1997) developed a reverse northern protocol in which radioactively-labeled cDNA synthesized from RNA samples are used as a probe and DD-cDNAs are fixed on a nylon membrane prior to cloning. Here we describe a modification using nonradioactive probes of the method of Zegzouti et al. (1997). This modified method is designed for use with reverse northern blots as a preliminary screening technique to analyze DD candidates. To illustrate this technique, we have carried out differential display reverse transcription (DDRT) using mRNA from embryogenic and nonembryogenic sugarcane calli and cell suspension cultures. Materials and Methods Plant material Experiments were carried out with sugarcane (Saccharum sp.) cv. V78-1 embryogenic and nonembryogenic calli obtained by the method of Ho and Vasil (1983). mRNA isolation mRNA isolation from callus and cell suspensions was performed with the PolyAtract® SYSTEM 1000 paramagnetic beads from PROMEGA following the manufacturer’s instructions. DDRT-PCR Conditions A total of 200 ng of mRNA isolated was reverse transcribed in a 20 µl reaction volume using 200 units of SuperScriptTM (Life Technologies, Eggenstein, Germany) and 5.5 M dT(18) (5’-biotin label) as a 3’-end primer to reverse transcribe the polyadenylated mRNAs. The conditions for reverse transcription were as described in the instruction manual for the SuperScriptTM enzyme, Gibco BRL. The concentration and integrity of cDNAs were evaluated by chemiluminescent detection. PCR amplifications were performed in 20 µl of a reaction mix with 1 µl of the first strand reaction, 2.5 M Oligo dT11(VN), 0.5 µM 10 mer OPERON primers, 50 M each dNTPs, 1x polymerase buffer [20 mM Tris-HCl (pH 8.4), 50 mM KCl], 1.5 mM MgCl2 and 1 unit of recombinant Taq DNA polymerase (GibcoBRL Life TechnologiesTM). PCR amplification reactions were initially incubated for 5 min at 94EC followed by 40 cycles at 94EC for 30 s, 42EC for 40 s, and 72EC for 1 min. An additional extension period at 72EC was programmed for 5 min. The resulting PCR products were analyzed in 1.4% agarose gels by electrophoresis and then stained with ethidium bromide. DNA fragments were visualized under UV light at 315 nm. Chemiluminescent detection was performed as specified by the supplier of Southern-LightTM nucleic acid detection system for biotin labeled probes (Tropix)
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with CSPD® substrate. Chemiluminescence of the blot was detected with standard x-ray film. Re-amplification, purification and blotting Weakly expressed DD bands were re-amplified by mixing the agarose gel containing DD-cDNA with 25 µl of distilled sterilized water, followed by centrifugation at 14,000 g in an Eppendorf 510 centrifuge for 20 min. After centrifugation, 5 µl of the DD-cDNA was used as a template for PCR amplifications using the same primers and conditions as for DDTR-PCR amplification in 50 µl. Bands of weak intensity were easily re-amplified under the same conditions as those described for amplification of the first strand cDNA, and the strong differentially expressed cDNA bands were purified directly using a QIAEXTM II kit (Quiagen, Hilden, Germany). The re-amplified DDRT-PCR bands (20 µl) were electrophoresed in 1.4% agarose gels, stained with ethidium bromide, visualized under UV light at 315 nm, and blotted on a Hybond-NX Amersham LIFE SCIENCE membrane using a conventional capillary procedure (Sambrook et al., 1989). After transfer, the membrane was air-dried and the DNA was fixed to the nylon membrane by baking at 80EC for 2 h. Reverse northerns Reverse northerns were performed following the procedure of Zegzouti et al. (1997), and modified as described below. Hybridization Duplicate membranes were pre-hybridized for 3 h in hybridization buffer (5x SSC, 0.1% (w/v) N-lauroylsarcosine, 0.02% SDS, 1% [w/v] blocking solution [1% blocking reagent, Boehringer Mannheim]). One membrane was hybridized with biotin labeled cDNA from embryogenic callus or cell suspensions, and the other was hybridized with cDNA from the corresponding nonembryogenic callus or cell suspensions. The probes were adjusted to a concentration of 25 ng/ml and hybridized for 16 h at 65EC. Results and Discussion Although total RNA is suitable for DDRT-PCR, using mRNA isolated with magnetic particles and streptavidin coupled with biotin labeled oligo dT(18) decreases the time for mRNA isolation and enhances the yield and purity. This procedure is most useful when quantity of starting materials is limited. Less manipulation of mRNA enhances the yield and obviates the need for DNase digestion of nucleic acid before RT, since DNA contamination is not likely to occur with this protocol. The most important features of our method are sensitivity and simplicity. One isolation of RNA from 100 mg of callus gives enough mRNA for expression analysis of many DD candidates in one hybridization without radioactivity risks or the laborious work of making northern blots for each candidate.
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Figure 1. Total cDNA labeled with biotin and electrophoresed in 5% polyacrylamide gels. EC: embryogenic callus; NEC: nonembryogenic callus.
cDNA labeling with biotin oligo dT(18) Detection with biotin labeled primers, used to observe DDRT-PCR products in polyacrylamide gels (An et al., 1996), produced less background than labeling probes by biotin incorporation of the modified nucleotide. The use of 5´ biotin labeled oligo dT(18) in the reverse transcription of total mRNA is an easy way to produce large amounts of labeled cDNAs. The biotinilated oligo dT(18) primer allowed us to analyze the integrity and relative concentration of cDNA (Figure 1). We observed that the amount of cDNAs in the different samples were similar, in the range of 100 - 3,000 bp. The strong signal observed under 100 bp corresponded to excess primer. These steps to check cDNA integrity and concentration are necessary to provide for consistent quality and signal strengths in reverse northern analyses. DD candidates blotting The capillary transfer procedure leads to very clean detection even after periods of over expression. Genes with very low expression could be detected over the high background without any interference (Figure 2). DD candidate detection Analysis of differential fragments by reverse northerns resulted in three types of DD fragment signals: (i) DD fragments that were not detected from either embryogenic or non embryogenic tissues, (ii) DD fragments that were detected in
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Figure 2. Nonradioactive reverse northern of different DD candidates. A: DD candidates in agarose gel (1.5%); B: Nonembryogeneic cDNA hybridized with DD candidates blotted on nylon membranes; C: Embryogeneic cDNA hybridized with DD candidates blotted on nylon membranes.
both cDNA categories, and (iii) differentially detected DD fragments such as that detected only with nonembriogenic calli cDNA probe in Figure 2. These classes of signals were also found with the radioactive detection method (Zegzouti et al. 1997). We were unable to detect many expected DD candidates, in accordance with other reports in which genes involved in early stages of somatic embryogenesis have very low expression (Heck et al., 1995) and are not detected by this technique. One of the DD genes we detected to be specific to nonembriogenic calli had a very low signal, only being recognized after 3 h of exposure. The rest of the DD candidates that we detected in both cDNAs had very strong signals after 15 min of exposure. In conclusion, these results demonstrate the advantage of screening DD candidates from DDRT-PCR using a nonradioactive detection system of biotin-streptavidin interaction coupled with alkaline phosphate and detection of the chemiluminescent CSPD® substrate. This procedure is a reasonable alternative to radioactive detection methods. Acknowledgements This work was supported by grants from Consejo de Desarrollo Científico y Humanístico CDCH-UCV to Maira Oropeza. References An G, Luo G, Veltri RW and O’Hara SM (1996) Sensitive, nonradioctive differential display method using chemiluminescent detection. BioTechniques 20: 342-346. Heck GR, Perry SE, Nichols KW and Fernández DE (1995) AGL15, a MADS domain protein expressed in developing embryos. Plant Cell 7: 1271-1282.
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Ho WJ and Vasil IK (1983) Somatic embryogenesis in sugarcane (Saccharum officinarum L.): the morphology and physiology of callus formation and the ontogeny of somatic embryos. Protoplasma 118:169-180. Liang P and Pardee AB (1992) Differential display of eukaryotic messenger RNA by means of the polymerase chain reaction. Science 257: 967-971. Liang P, Averboukh L and Pardee AB (1993) Distribution and cloning of eukaryotic mRNA by means of the differential display: refinements and optimization. Nucleic Acids Res 21: 3269-3275. Lohmann J, Schickle H and Bosch T (1995) REN Display, a rapid and efficient method for nonradiactive differential display and mRNA isolation. BioTechniques. 18: 200-202. Luehersen KR, Marr LL, Van der Knaap E and Cumberledge S (1997) Analysis of differential display RT-PCR products using fluorescent primers and GENESCANTM software. BioTechniques 22: 168-174. Rompf R and Kahl G (1997) mRNA differential display in agarose gels. BioTechniques 23: 28-32. Sambrook J, Fritsch EF, Maniatis T (1989) Analysis of Genomic DNA by Southern hybridization. In: Molecular Cloning (a laboratory manual), pp. 9-31. Cold Spring Harbor Laboratory Press. Second Edition. Zegzouti H, Marty Ch, Jones B, Bouquin T, Latché A, Pech JC and Bouzayen M (1997) Improved screening of cDNA generated by mRNA differential display enables the selection of true positives and the isolation of weakly expressed messages. Plant Mol Biol Reptr. 15: 236-245.
