Analysis of Gene Expression in Striatal Tissue by Multiprobe RNase Protection Assay - Drugs of Abuse: Neurological Reviews and Protocols - page 178

mRNA and protein levels in evaluating regulation of protein function therefore
cannot be overestimated.
The major advantage of Northern blot analysis lies in the ease of the
technique, which is generally less problematic than any of the other methods.
The major disadvantages of Northern blot lie in the lack of sensitivity and
the lack of anatomic specifi city. The major advantage of in situ hybridization
lies in the anatomic specifi city provided. This is of particular importance for
studies in the CNS, in which discrete populations of cells within a given brain
region may play an important regulatory role. The disadvantage of in situ
hybridization lies in the lower sensitivity compared to RT-PCR, and the lack of
absolute specifi city when studying closely homologous mRNAs. In contrast,
the advantage of RT-PCR lies in the exquisite sensitivity resulting from the
multiple amplifi cation steps of this method.
One of the major advantages of the RNase protection assay is the absolute
specifi city of this technique in determining expression of the mRNA of interest.
This advantage is particularly powerful in studies examining expression of
closely homologous transcripts, or of alternatively spliced transcripts arising
from a single gene. Second, RNase protection assay is highly sensitive. In
practice, it is possible to detect reliably approx 1-5 pg of target RNA with
the RNase protection assay employing high specifi c activity probes. The third
major advantage of this technique is the ease with which multiple samples may
be assayed, once a given assay is established. This makes it feasible to measure
mRNA expression in individual animals from several reasonably sized groups,
an approach frequently employed in studies simultaneously measuring the
behavioral and cellular effects of drugs of abuse (3 , 4) .
2. Materials
2.1. cDNA Insert Plasmid Construction
Plasmids used for synthesis of both synthetic RNA standards used for
standard curves and cRNA probes may be obtained by synthesizing your own
( see Subheading 3.3. ), or “clone-by-phone.” It's generally worth the effort
it takes to send an e-mail to try and obtain an existing clone from another
researcher or commercial source prior to embarking on your own synthesis. If
synthesized in-house, the following materials are needed.
1. M-MLV reverse transcriptase (Invitrogen).
2. Restriction enzymes (Invitrogen).
3. Yeast tRNA (RNase-free, Invitrogen cat. no. 15401-011).
4. Random hexamers (Invitrogen).
5. Oligo(dT)-cellulose spin columns (Clontech).
6. pGEM-4 Vector and RNasin (Promega).