Ion Channel Assessment - Animal Models of Acute Neurological Injuries: Injury and Mechanistic Assessments - page 506

3.10. Ion Channel
Assessment:
Two Exemplary Key
Methods

In particular, the wide-spread spectrum of methods evaluation ion
channels cannot be discussed here. Therefore, principles of two key
methods are highlighted. Transcript analysis is one key method.
Northern blot analysis is usually displaced by RT of isolated RNA
and following PCR. Prerequisite is isolation of RNA. Here, an easy
way to isolate RNA from tissues or cultivated cells is a single-step
method based on acid guanidinium thiocyanate-phenol-chloroform
extraction described by Chomczynski and Sacchi ( 56 ). Then, RNA
has to be transcribed into DNA by reverse transcription, followed
by standard PCR allowing an amplifi cation of the cDNA. Here, a
careful selection of the primer pair is important. Primer pairs should
be highly selective and sensitive for the individual particular chan-
nel, but for nearly all interesting species and channel subtypes char-
acteristic primer pairs have been described in literature. Intern
controls, like amplifi cation of housekeepers transcript (e.g., -actin-

or hypoxanthine phosphoribosyltransferase-HPRT) or control
samples without reverse transcriptase, enable to value the quality
and quantity of amplifi ed nucleic acids and to detect contamina-
tion. Different variations of standard RT-PCR are elegant method
to answer special questions: While standard PCR allows at best
only a semi-quantitative analysis, real-time PCR enables a quantifi -
cation of the amplifi ed DNA. Transcript analysis of single isolated
cells is possible with single-cell PCR analysis. Here, commercial
and easy-to-handle kits are available.
Besides transcript analysis by RT-PCR, direct protein detection
by Western-blot analysis is the molecular-biological standard in
assessing ion channel expression. This method is based on isolation
of microsomes, separation of the proteins according to their elec-
trophoretic mobility under denaturating conditions by sodium
dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE),
transfer of the proteins on a membrane (e.g., nitrocellulose or
polyvinylidene fl uoride membrane) and following detection of the
Ca 2+ channels. Preparation of microsomes can easily be performed
with a modifi ed protocol described by Flockerzi et al. ( 57 ). For
SDS-PAGE, electrophoretic separation of high molecular proteins
like calcium channels with a molecular weight between 212 and
273 kDa depending on the Ca v subunit require low concentrated
separation gels (about 5-7.5%). Low concentrated separation gels
lead to diffuse lanes whereas protein transfer is more diffi cult from
higher concentrated separation gels on the membrane. The most-
common transfer method for proteins is Western-blotting, which
in principle can be performed with the tank-blot or the semidry
blotting system. Successful performance of Western-blot analysis
requires an optimization of several parameters, including adaption
of the temperature, pH, preparation of the membrane and samples
and used puffer systems, especially of the concentration of metha-
nol advancing resolution of the blot and SDS carrying elution of
proteins from polyacrylamide gel and inhibiting precipitation

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