Analysis of mRNA Expression in Striatal Tissue by Differential Display Polymerase Chain Reaction - Drugs of Abuse: Neurological Reviews and Protocols - page 201

3.7. After DDPCR
Once differential expression has been confi rmed, sequence the cDNA if you
have not already done so (well-isolated cDNAs can be directly cycle sequenced
using the T7, SP6 sites or custom primers; alternatively, subclone and obtain
minipreps as described earlier). Isolate the portion of the sequence that is
not vector or primer and compare it to a public resource such as GenBank
(www.ncbi.nlm.nih.gov). The cDNA may match a gene that is already known
to have differential expression (from other experiments or because it already
showed up earlier in the DDPCR experiment). In such cases, be sure to check
carefully that the sequences match along their full length—DDPCR can turn
up novel or unexpected splice variants that may have important functional
roles. For this reason it is also a good idea to perform a Northern blot, with an
RNA ladder (e.g., Ambion), to establish the full-length size of mRNAs whose
differential expression has been confi rmed ( see Note 24 ). If the gene is novel or
poorly characterized, the next step is to obtain the sequence of the full-length
mRNA sequence. This can often be obtained directly from an established
GenBank entry; if not, often the sequence can be inferred by assembly from
many GenBank EST (expressed sequence tag) sequences. Barring that, if the
genomic sequence is known, a number of PCR primers can be designed and
used for RT-PCR in combination with PCR primers antisense to the 5′ end
of the DDPCR sequence (this is a quick but hit-and-miss approach). Finally,
the remainder of the mRNA sequence can be obtained “the hard way”—by
performing rapid amplifi cation of cDNA ends (RACE; e.g., 6 ) or by screening
cDNA libraries for clones that contain the DDPCR fragment ( see Note 25 ).

4. Notes

1. It is only the bases at the 3 end of the primer that participate in the annealing

to the cDNA population. Some investigators have found that it is desirable to
have C or G bases, which form more stable bonds than A or T bases, in the most
3 one or two positions.

2. It is essential to avoid RNase contamination during the initial stages of the proce-
dure, and widespread DNA contamination thereafter. If you are already routinely
performing successful Northern blots, your materials-handling techniques are
probably fi ne. Otherwise, follow the following steps. Keep all compounds, fl uids,
and so forth used in RNA handling separate from other lab materials and handle
them only while wearing gloves. Any solutions, buffers, and so forth should
either be prepurchased in sealed, RNase-free form, or made up with diethyl
pyrocarbonate (DEPC)-treated H 2 O. The source of water used in RT-PCR is
particularly important—unfortunately, nuclease-free water provided by different
manufacturers can give variable results. Find a water source that works and
use it consistently. Use RNase-free pipet tips with an aerosol blocking fi lter.