A modification of Representational Difference Analysis, with application to the cloning of a candidate in the Reelin signalling pathway
© Kuvbachieva and Goffinet; licensee BioMed Central Ltd. 2002
Received: 28 January 2002
Accepted: 24 April 2002
Published: 24 April 2002
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.
In the post-hybridization mix, the 5'-ends of homoduplexes of interest (tester-tester) are filled-in with α-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.
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.
Isolation of NAM16 using a modification in the cDNA-RDA protocol
As other PCR-based screening techniques, RDA can result in amplification bias and generate spurious clones that do not correspond to differentially expressed genes. In our experiment, following the classical protocol, a high number of gene fragments were not differentially expressed. Furthermore, the basic RDA protocol is rather time consuming and labour intensive. In addition to enriching the population in differential tester fragments, the final PCR of each RDA round also yields unwanted products. Among others, this is due to: i) lack of sufficient driver competition in the formation of the tester homoduplexes during hybridization, especially from abundant tester samples; ii) the linear increase in contaminating heteroduplexes; iii) inefficient annealing in the complex mixture after the subtraction step; and iv) trapping of a fraction of the tester molecules in heteroduplexes. All these factors may contribute to increasing the non-specific amplification of spurious fragments that do not represent genes with differential expression between the tester and the driver. In consequence, multiple iterations of the enrichment procedure are usually required in order to increase the proportion of target homoduplex sequences. As a partial solution to these inconveniences, we propose a modification that seems able to reduce the complexity of the amplification mixture, thus rendering cDNA-RDA procedures more rapid and user friendly. By following this modified protocol, we were able to clone a fragment of a novel gene that is differentially expressed in normal and Dab1-deficient mRNA and is thus a potential new member of the reelin signalling pathway.
Applying α-thio-deoxynucleotides in the tester preparation for the cDNA-RDA protocol allows to protect tester heteroduplexes in the post-hybridization mixture from the enzymatic degradation of ExoIII. This modification of the original RDA protocol  may reduce the background due to amplified products deriving from false positive fragments, thus reducing significantly the complexity of the post-hybridization mix. In addition, multiple rounds of subtraction and amplification are avoided and the protocol is rendered less time-consuming. This is illustrated by the cloning of Nam16, a novel gene differentially expressed in the brain of normal and Dab1-deficient mice.
Materials and Methods
Representational Difference Analysis
Normal BALB/c, reelin -/- and Scrambler (Dab1-/-) mice were bred as described . All animal procedures were approved by the institutional Animal Ethics Committee. Brains from four to six newborn (P0) animals were dissected under cold anesthesia and total RNA extracted using an RNeasy RNA kit (Qiagen). Poly(A)+-RNA was prepared with PolyATract mRNA Isolation Systems (Promega). cDNA was synthesised by oligo(dT) priming using Superscript II as recommended by the manufacturer (Invitrogen). Double-stranded cDNA was synthesised from 5 micrograms poly(A)+-RNA and RDA was performed as described by Hubank and Schatz . Restriction reactions were performed with DpnII (New England Biolabs). PCR amplification was carried out using Taq DNA polymerase (Invitrogen). For fill-in reactions, Klenow Fragment (Promega) was added at the proportion of 1 Unit per μg DNA, together with dNTPs (Roche), at a final concentration of 40 μM, and the mixture was incubated for 30 minutes at 37°C for 30 minutes, followed by heating for 10 min at 70°C. Conditions for fill-in with α-thio-dNTP (Promega) were similar. After fill-in, the mixture was submitted to two rounds of extraction with phenol:chlorophorm (1:1) and precipitated with ethanol in the presence of 5 micrograms of glycogen. Digestion was performed for 35 min at 37°C with 300 U of Exonuclease III (Promega) and 10 U of Mung bean nuclease (New England Biolabs) in the presence of 20 mM Tris-acetate, 10 mM Mg++-acetate, 50 mM K+-acetate, 1 mM DTT, 0,05 mg/ml BSA. To inactivate the enzymes 4 reaction volumes of 50 mM Tris. HCl (pH8,9) were added and the samples were incubated for 10 min at 75°C. This was followed by 28 cycles of PCR following the conditions described in . The final difference products were cloned into the pCR2.1 vector (Invitrogen), and sequenced with an ABI prism sequencer using AmpliTAq DNA Polymerase and a Big Dye Terminator Cycle sequencing kit (PE Biosystems, England).
Northern blot analysis
2,5 μg of poly (A) RNA were electrophoresed in an agarose-formaldehyde gel and blotted to Hybond N nylon membranes (Amersham). After UV-crosslinking, membranes were hybridised with [32P]-dCTP (ICN Biomedicals NV/SA) labelled DNA probes prepared with the RadPrime DNA Labeling System (Invitrogen). After overnight hybridization at 42°C in 50% formamide , the membranes were washed twice in 2 × SSC/0,1% SDS for 15 min at room temperature and then once in 1 × SSC/ 0,1% SDS for 15 min at 42°C. mRNA sizes were estimated by comparison with an RNA ladder (Invitrogen). For quantitative analysis, the signals on the membranes were quantified using a Cyclone (Packard) apparatus and the OptiQuant program (Fig. 4).
We wish to thank D. Schatz and J. C. Renauld for communication of RDA protocols and advice, and C. Dernoncourt for technical assistance. This work was supported by the Fondation médicale Reine Elisabeth, the Actions de Recherches Concertées (grant 98-02/186) and grant FRFC 2.4504.01.
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