Characterization of RNA - Animal Models of Acute Neurological Injuries: Injury and Mechanistic Assessments - page 149

normal tissue. The C t values of both the control and the samples of
interest are normalized to an appropriate endogenous housekeep-
ing gene ( 58 ).
It is similar to the relative standard curve method, except
arithmetic formulas are used to achieve the result for relative
quantitation:

Δ Δ C t = Δ C T , sample − Δ C t , reference,

when

C T , sample = the C t value for any sample normalized to inter-
nal standard (endogenous housekeeping gene).

Δ

C t , reference = the C t value for the calibrator also normalized
to internal standard.
For the

Δ

C t calculation to be valid, the amplifi cation effi cien-
cies of the target and the internal standard endogenous reference
must be approximately equal. This can be established determining
the variance of the D C t ( C T target − C T internal standard) with tem-
plate dilution. If effi ciency of amplifi cation of the target gene and
internal standard are the same, D C t will be equal for all template
dilutions. However, if the amplifi cation effi ciency of the target
gene and internal standard are not equal, this may indicate compe-
tition in the multiplex reaction. If this is the case, a new internal
standard should be selected. (Recommend serial fi vefold dilutions
of original cDNA template).

Δ Δ

4. Conclusion

Motivated by the progress in understanding the role of regulatory
variants in human phenotypic variation, gene expression has
become an obvious candidate as the informative intermediate of
the “DNA phenotype.” The emergence of high-throughput tech-
nologies, such as the Affymetrix GeneChip and the Illumina
BeadChip, have fueled in-depth studies of the human transcrip-
tome and next generation sequencing technologies are only
expected to catapult this interest. Given the diffi culty of studying
gene expression profi les of human brain, it will be important to
continue to study the effects of brain gene expression in models of
animal brain injury, making comparisons between brain and blood
gene profi les that can be understood in the context of human
disease.
The characterization of mRNA via gene expression analysis
provides novel candidates for the study of brain injury, however,
tells us little about translational or posttranslation events that
relate to protein expression and thus the true response of cells
to brain injury and neurological disease. Therefore, a variety of