Analysis of mRNA Expression in Striatal Tissue by Differential Display Polymerase Chain Reaction - Drugs of Abuse: Neurological Reviews and Protocols - page 200

desired, the accuracy of the band cut can be checked by reexposing the remainder
of dried gel to fi lm.

3.5. Reamplifi cation

1. Reamplifi cation reaction components are as follows ( see Note 19 ):

H 2 O 21.2 µ L

10X Expand HiFi standard buffer 1 4.0 µ L

dNTP (250 µ M ) 1 6.4 µ L

Upstream reamp primer (2 µ M ) 1 4.0 µ L

Downstream reamp primer (2 µ M ) 1 4.0 µ L

Expand High Fidelity polymerase 1 0.4 µ L

.. (40 µ L total per reaction)

2. The reamplifi cation primers are designed to work with bands generated using
any combination of DDPCR primers. Therefore a single mix suffi ces for all
reamplifi cation reactions. Add 10% extra to compensate for pipetting losses. Add
40 L of this mix directly into each PCR tube containing a dried gel fragment

and place in a preheated thermal cycler. Start the following program: 95°C,
2 min; 40 cycles of {92°C, 15 s; 60°C, 5 s, 72°C, 2 min + 2 s/cycle}; 68°C,
7 min; 4°C, hold.

µ

3. When completed, add 10 L of 5X DNA loading dye to each of the PCR tubes,

and mix by pipetting up and down. Run 20

µ

L from each tube on a standard 1%
agarose-EtBr gel, including a (nonradioactive) DNA ladder in a spare lane. Take
a picture of the resulting bands on a UV light box, and compare their sizes to
those on the DDPCR gel. Cut out the succesfully reamplifi ed bands and purify
the DNA ( see Notes 20 and 21 ).

µ

3.6. Confi rming Differential Expression
At this stage, the DNA can be directly used as a template for generating
an antisense RNA probe (by in vitro transcription using T7 polymerase) and
differential expression confi rmed by in situ hybridization, using standard
protocols. This is the preferred approach if your DDPCR fi lms show signifi cant
separation between bands. If the DDPCR bands are packed tightly together, it
will be hard to cut out a single band cleanly (and a single position on the gel may
well contain a mixture of cDNA species). In this situation, subclone the reampli-
fi ed DNA by ligation into a standard vector with antibiotic resistance ( see
Note 22 ), transform competent cells, grow on an agar/antibiotic plate overnight,
pick colonies (eight colonies is usually enough), grow up a few milliliters
of culture overnight, and perform minipreps. If you have access to a mass-
sequencing facility, sequence all the minipreps; otherwise try using the fi rst one
to confi rm differential expression and proceed from there ( see Note 23 ).