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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.


Schematic representation of the results from quantitative Northern blot analysis All hybridization signals have been normalised with the signal from beta-actin and estimated using the Cyclone PhosphorImager. Details are described in the text. The bar represents the variation of two duplicate experiments.
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Figure 4: Schematic representation of the results from quantitative Northern blot analysis All hybridization signals have been normalised with the signal from beta-actin and estimated using the Cyclone PhosphorImager. Details are described in the text. The bar represents the variation of two duplicate experiments.

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)

Schematic representation of the results from quantitative Northern blot analysis All hybridization signals have been normalised with the signal from beta-actin and estimated using the Cyclone PhosphorImager. Details are described in the text. The bar represents the variation of two duplicate experiments.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC113762&req=5

Figure 4: Schematic representation of the results from quantitative Northern blot analysis All hybridization signals have been normalised with the signal from beta-actin and estimated using the Cyclone PhosphorImager. Details are described in the text. The bar represents the variation of two duplicate experiments.
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.