Identifying differentially expressed cDNAs from mixed cDNA clones

If a DDRT band excised from a gel contains contaminating cDNAs (cDNAs of the same size that are co-migrating through the gel), multiple mRNAs will be identified on a Northern blot and it will be impossible to discern which o f these mRNAs represent the differentially expressed cDNA that was originally identified. These contaminating sequences may originate from DNA present in the RNA sample used for reverse transcription, cDNAs that are co-migrating or cDNAs that were co-purified by mistake when the band was excised. It is therefore important to separate these cDNAs before characterization. As mentioned, direct sequencing o f the DDRT fragment will show whether the excised band contains a mixture of clones. If the fi-agment is first cloned into a vector, multiple cDNAs will be cloned separately and sequencing only one o f these clones may not provide information on the actual cDNA that is showing differential expression.

3.7.4.1 Reverse Northerns

Methods for dot-blot screening o f large numbers o f DDRT products have been described: RNA used for the original DDRT reaction is reverse transcribed, using a standard oligo-dT primer and radiolabelled by random priming. Plasmid DNA is prepared from cloned DDRT products by mini-preparation and dot-blotted onto filters in duplicate; or alternatively, DDRT products that have been re-amplified by PCR are denatured and directly applied to the filters. The filters are then hybridized separately to each of the radiolabelled cDNA probes prepared from the starter RNA populations. It is important that each of the probes is radiolabelled to the same specific activity so that differences seen in the intensity o f hybridization between samples can be correlated with level o f expression. The same protocol can be used for differential screening by colony hybridization. In this case, colonies o f cloned DDRT-PCR products are grown overnight and then replica-plated onto duplicate membranes. These are then denatured and neutralized and the membranes then hybridized with the same radiolabelled probes used for the dot blots.

With this method it is therefore possible to confirm the differential expression of a large number of excised DDRT products (Mou et a l, 1994, Zhang et a l, 1996).

Vogeli-Lange et a l (1996) have proposed a similar protocol where an aliquot of

the original DDRT-PCR reaction carried out in the presence o f ^^P-dATP is hybridized to dot-blotted DDRT-PCR products.

3.T.4.2 Reverse Southerns

This method involves the electrophoresis o f cloned DDRT-PCR products through duplicate agarose gels. If 5-10 clones for each band excised and eluted from the DDRT gel are analyzed, this should ensure that the clone containing the differentially expressed sequence will be identified. The agarose gels are then Southern blotted and hybridized with radiolabelled cDNA probes prepared from the same RNA used in the original DDRT reaction. Consalez et a l (1996) suggest that this method is quantitative and can detect mRNAs that have a low expression.

Alternatively, a re-run o f the original differential display gel can be Southern blotted and this can be probed with a radiolabelled clone to confirm that the clone does indeed contain the differentially expressed sequence (McClelland et a l, 1995).

3.7.4.3 Northern blot affinity capture

This technique involves hybridizing DDRT fragments to Northern blots and recovering the mRNAs showing differential expression directly from the blot. This means that if the fragment did contain co-migrating cDNAs that are not differentially expressed, they will not be selected. The piece of Northern blot is stripped by boiling in water and the DNA recovered by ethanol precipitation and re-amplified by PCR. After cloning, the insert should be used to probe another Northern blot to confirm the same result as before

{ U e t a l , 1994).

3.7.4.4 Separation of DDRT fragments through Single-Stranded Conformation Polymorphism (SSCP) gels

This method can be used to separate cDNA products o f a similar size which have different sequences and is usually used to differentiate between sequences, one o f which is thought to contain a point mutation (Hayashi, 1991). The principle will still work for DDRT-PCR products and can be used to separate mixed products from a single excised band (Mathieu-Daude, 1996). The differentially displayed cDNA can be identified if a corresponding DDRT band is excised from one of the DDRT lanes not containing the tissue/cell type of interest and is simultaneously electrophoresed through the SSCP gel. (It must be noted that each PCR product can be expected to produce two bands in an SSCP gel, corresponding to the two cDNA strands as the products will be denatured).

3.7.4.5 Electrophoresis through agarose gels containing bisbenzimide/PEG dye

This method can be used to separate mixed cDNA clones o f the same size but of different sequence (Wawer et a l, 1995) and has been used successfully to separate DDRT-PCR products (Geisinger et a l, 1997).

The DNA ligand bisbenzimide, when covalently coupled to polyethylene glycol (PEG) 6000, will preferentially bind to A+T sequence motifs in DNA. Thus, these sequences will consequently electrophorese slower through agarose gels that contains this dye. This allows for separation o f mixtures o f products that cannot be differentiated by size, but vary in sequence. Sequences up to 1 SOObp have been resolved by bisbenzimide/PEG, a resolution that cannot be achieved by SSCP gels (Wawer et a l ,

1995). This method is preferable to previously mentioned methods as it does not require the use o f radioactivity (the gels are stained with ethidium bromide) and is much quicker.

3.T.4.6 Restriction enzyme fingerprints of excised DDRT bands

A method has been suggested to identify the differentially expressed fragment from several similar sized ones which involves the generation of a fluorescent restriction enzyme fingerprint from the excised band, a supposed mixture o f cDNAs. cDNA subclones derived from this band will contain sequences of similar size but of different sequence to the actual differentially expressed cDNA. If restriction enzyme fingerprints are also generated from these subclones, then those that match the most intense fingerprint pattern seen in that from the excised band will represent the differentially expressed cDNA (Smith et a l, 1997).