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A modification of Representational Difference Analysis, with application to the cloning of a candidate in the Reelin signalling pathway.

Kuvbachieva AA, Goffinet AM - BMC Mol. Biol. (2002)

Bottom Line: In the post-hybridization mix, the 5'-ends of homoduplexes of interest (tester-tester) are filled-in with alpha-thio-deoxynucleotides.Unprotected duplexes, as well as the single-stranded DNA fragments, are degraded using ExoIII and Mung Bean Nuclease, prior to PCR subtraction, resulting in less complex difference products.We propose a modification of cDNA-RDA that may reduce the complexity of the post-hybridization mix and thus facilitate the amplification of differentially expressed products.

View Article: PubMed Central - HTML - PubMed

Affiliation: Neurobiology Unit 2853, Univ. Namur Med. Sch., 61, rue de Bruxelles, Namur, Belgium. kuv1302@yahoo.co.uk

ABSTRACT

Background: cDNA-RDA is one of the subtractive cloning techniques used to isolate differentially expressed genes between two complex cDNA populations. In the present study we present a modification of the protocol described by Hubank and Schatz.

Results: In the post-hybridization mix, the 5'-ends of homoduplexes of interest (tester-tester) are filled-in with alpha-thio-deoxynucleotides. Unprotected duplexes, as well as the single-stranded DNA fragments, are degraded using ExoIII and Mung Bean Nuclease, prior to PCR subtraction, resulting in less complex difference products. We illustrate this modification by the cloning of a new gene which is differentially expressed in normal, reelin and Dab1 mutant mice and is a candidate member of the Reelin signalling pathway involved in brain development.

Conclusion: We propose a modification of cDNA-RDA that may reduce the complexity of the post-hybridization mix and thus facilitate the amplification of differentially expressed products.

No MeSH data available.


Northern blot-analysis using NAM16 as probe. 2.5 microgram of poly(A)+-RNAs from normal newborn (P0) brains were electrophoresed, blotted to Hybond N nylon membranes (Amersham), and hybridized with α32P-labeled DNA probes.
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Figure 3: Northern blot-analysis using NAM16 as probe. 2.5 microgram of poly(A)+-RNAs from normal newborn (P0) brains were electrophoresed, blotted to Hybond N nylon membranes (Amersham), and hybridized with α32P-labeled DNA probes.

Mentions: We were interested in applying RDA to examine how the extracellular protein Reelin influences the expression of specific genes during brain development. For the subtractive hybridization, a representation from normal newborn mice brains was used as the driver. The tester derived from scrambler (mDab1-deficient) brains at the same developmental stage. Whereas the representations showed a typical smear in gel electrophoresis (Fig. 2A), the product of the first round of the modified RDA protocol had reduced complexity, with only a few prominent bands (Fig. 2B). In contrast, in a parallel subtraction carried out with the same tester and driver DNA preparations by following the classical protocol of Hubank and Schatz, a difference product showing a few discrete bands was obtained only after the second round of enrichment (DP-2), as shown in Fig. 2D. The whole products DP-1 (Fig. 2C) and DP-2 (Fig. 2D) were cloned into pCR2.1 (Invitrogen). Six and 8 different cDNA fragments were obtained respectively, from DP-1 and DP-2. None of the 8 DP-2 fragments was differentially expressed. Among the six DP-1 fragments, two were from known mouse genes that code for proteins involved in general cellular functions (alpha-tubulin (Tuba1)) and DNA-metabolism (M-phase phospho-protein); one was highly similar to a novel human gene and three had partial homology to mouse EST. Among the novel fragments cloned, a partial 530 bp long cDNA could be extended to 1146 bp by using alignment with genomic and EST databases, and verification by RT-PCR. Northern blot analysis revealed that this gene is expressed in the brain as an approximately 6.5 kb mRNA (Fig. 3). Duplicate quantitative Northern blots analysis of normal (N), reelin-deficient and mDab1-deficient (scrambler) mRNA was carried out by hybridising blots sequentially with the radiolabeled candidate fragment and with a control beta-actin probe. The signals were estimated using a Cyclone-Phosphor Imager apparatus and the OptiQuant program. As shown in Fig. 4, the expression level of this new gene is comparable in normal and reelin-deficient brains but is reduced by about 50% in Dab1deficient brains. This observation of reduced expression in the Dab1-mutant mouse brain suggests that this novel gene is a potential partner in the Reelin-signalling pathway implicated in brain development. Because the database searches revealed that this fragment did not match any known entry, it was provisionally named Nam16. The full-length message and its characterization are currently under investigation.


A modification of Representational Difference Analysis, with application to the cloning of a candidate in the Reelin signalling pathway.

Kuvbachieva AA, Goffinet AM - BMC Mol. Biol. (2002)

Northern blot-analysis using NAM16 as probe. 2.5 microgram of poly(A)+-RNAs from normal newborn (P0) brains were electrophoresed, blotted to Hybond N nylon membranes (Amersham), and hybridized with α32P-labeled DNA probes.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC113762&req=5

Figure 3: Northern blot-analysis using NAM16 as probe. 2.5 microgram of poly(A)+-RNAs from normal newborn (P0) brains were electrophoresed, blotted to Hybond N nylon membranes (Amersham), and hybridized with α32P-labeled DNA probes.
Mentions: We were interested in applying RDA to examine how the extracellular protein Reelin influences the expression of specific genes during brain development. For the subtractive hybridization, a representation from normal newborn mice brains was used as the driver. The tester derived from scrambler (mDab1-deficient) brains at the same developmental stage. Whereas the representations showed a typical smear in gel electrophoresis (Fig. 2A), the product of the first round of the modified RDA protocol had reduced complexity, with only a few prominent bands (Fig. 2B). In contrast, in a parallel subtraction carried out with the same tester and driver DNA preparations by following the classical protocol of Hubank and Schatz, a difference product showing a few discrete bands was obtained only after the second round of enrichment (DP-2), as shown in Fig. 2D. The whole products DP-1 (Fig. 2C) and DP-2 (Fig. 2D) were cloned into pCR2.1 (Invitrogen). Six and 8 different cDNA fragments were obtained respectively, from DP-1 and DP-2. None of the 8 DP-2 fragments was differentially expressed. Among the six DP-1 fragments, two were from known mouse genes that code for proteins involved in general cellular functions (alpha-tubulin (Tuba1)) and DNA-metabolism (M-phase phospho-protein); one was highly similar to a novel human gene and three had partial homology to mouse EST. Among the novel fragments cloned, a partial 530 bp long cDNA could be extended to 1146 bp by using alignment with genomic and EST databases, and verification by RT-PCR. Northern blot analysis revealed that this gene is expressed in the brain as an approximately 6.5 kb mRNA (Fig. 3). Duplicate quantitative Northern blots analysis of normal (N), reelin-deficient and mDab1-deficient (scrambler) mRNA was carried out by hybridising blots sequentially with the radiolabeled candidate fragment and with a control beta-actin probe. The signals were estimated using a Cyclone-Phosphor Imager apparatus and the OptiQuant program. As shown in Fig. 4, the expression level of this new gene is comparable in normal and reelin-deficient brains but is reduced by about 50% in Dab1deficient brains. This observation of reduced expression in the Dab1-mutant mouse brain suggests that this novel gene is a potential partner in the Reelin-signalling pathway implicated in brain development. Because the database searches revealed that this fragment did not match any known entry, it was provisionally named Nam16. The full-length message and its characterization are currently under investigation.

Bottom Line: In the post-hybridization mix, the 5'-ends of homoduplexes of interest (tester-tester) are filled-in with alpha-thio-deoxynucleotides.Unprotected duplexes, as well as the single-stranded DNA fragments, are degraded using ExoIII and Mung Bean Nuclease, prior to PCR subtraction, resulting in less complex difference products.We propose a modification of cDNA-RDA that may reduce the complexity of the post-hybridization mix and thus facilitate the amplification of differentially expressed products.

View Article: PubMed Central - HTML - PubMed

Affiliation: Neurobiology Unit 2853, Univ. Namur Med. Sch., 61, rue de Bruxelles, Namur, Belgium. kuv1302@yahoo.co.uk

ABSTRACT

Background: cDNA-RDA is one of the subtractive cloning techniques used to isolate differentially expressed genes between two complex cDNA populations. In the present study we present a modification of the protocol described by Hubank and Schatz.

Results: In the post-hybridization mix, the 5'-ends of homoduplexes of interest (tester-tester) are filled-in with alpha-thio-deoxynucleotides. Unprotected duplexes, as well as the single-stranded DNA fragments, are degraded using ExoIII and Mung Bean Nuclease, prior to PCR subtraction, resulting in less complex difference products. We illustrate this modification by the cloning of a new gene which is differentially expressed in normal, reelin and Dab1 mutant mice and is a candidate member of the Reelin signalling pathway involved in brain development.

Conclusion: We propose a modification of cDNA-RDA that may reduce the complexity of the post-hybridization mix and thus facilitate the amplification of differentially expressed products.

No MeSH data available.