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

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5. It is usually most convenient to use a full ice bucket as a work surface. Many

kinds of pipet tips come prearrayed in microtiter racks; the tops of these racks

can be detached and used to keep strips of 0.2-mL tubes or 96-well PCR plates

chilled on the ice surface. If you have a thermal cycler that accepts 0.2-mL tubes,

it can be convenient to assemble the RT reactions in strips of 8

×

0.2 mL attached

tubes with individual lids.

6. Remember to make enough mix to compensate for pipetting losses; for example,

if using three RT primers and eight comparison animals (24 combinations), make

up a 27-reaction mix.

7. It is also possible to try slightly higher temperatures than 37°C toward the end

of the RT reaction. At higher temperatures (e.g., 42 or 45°C) the enzyme will

be degraded faster but there will be less secondary structure in the mRNA; such

secondary structure may impede the enzyme and cause some cDNA transcripts

to be truncated. At the end of the RT reaction some protocols call for a brief

incubation with RNase H, to remove the RNA from the newly formed cDNA.

However, omitting this step does not seem to make a substantial difference.

8. It is usually convenient to make up one “supermix,” which lacks primers, and

split it into submix tubes to which specifi c primers have been added.

9. If you're only comparing two conditions with two animals/condition, you could

use four upstream primers/96-well plate (each upstream primer would be used

in a 4

6 well quadrant).

10. Remember to mark, snip, or otherwise indicate which is the top left corner of the

PCR plate; otherwise you may turn it through 180° by mistake.

11. 33 P is vastly superior to 35 S for DDPCR. 35 S requires longer gel exposure times,

but more importantly forms volatile products during DDPCR that contaminate

the thermal cycler and potentially the rest of the laboratory.

12. With a large number of components to be added to specifi c combinations of PCR

tubes, it is easy to make a time-consuming and expensive mistake—for example,

adding RT reactions to the eighth column when the seventh was intended. To

help avoid this, it helps to use a marker pen to divide the PCR plate visually

into halves or thirds. Starting with a fresh array of 96 pipet tips and using the

missing tips as a visual guide to which corresponding tubes have been already

worked on is also useful.

13. This protocol is designed to generate DDPCR products of up to 2 kb, which

can be resolved on a Genomyx sequencer; if using a conventional metal-backed

sequencing gel apparatus, the polymerase extension times (the 72°C periods)

can be substantially shortened.

14. It is a very good idea to label radioactively a DNA ladder (e.g., 100-basepair

(bp) ladder from Gibco) by standard methods (e.g., T4 polynucleotide kinase

with [

×

- 33 P]ATP) and run it alongside the DDPCR products. This will allow a

reasonable estimate of band size, so that problems with reamplifi cation can be