Whole Cells - Molecular Theory of the Living Cell - page 392

where symbolizes a proportionality and K is a function, denoted as f in Eq. 12.4 ,

of at least eight parameters, denoted as P i , each reflecting the characteristics of
one of the eight arrows or Steps 1-8, in Fig. 12.5 :

1

K ¼ f(P i Þ (12.4)

where the index i runs from 1 to 8.
Combining Eqs. 12.3 with 12.4 leads to:

S ¼ f(P i Þ½ R C (12.5)

Equation 12.5 indicates that the microarray signal S would be proportional to the
intracellular RNA levels, if and only if all the P i values remain constant where the
index i runs from 1 to 8. As evident in Fig. 12.5 , the eight parameters that connect
R C to S can be divided into two groups - (1) the biological parameters ,P 1 ,P 2 ,P 3 ,
P 7 , and P 8 , and (2) what may be called the measurement parameters ,P 4 ,P 5 , and P 6 .
The reproducibility of the measurement parameters from one experiment to another
can be readily gauged by repeating a measurement three or more times using the
same biological samples. The accuracy and reproducibility of the DNA microarray
technique has been improving since its invention in the mid-1990s so that, under
ideal conditions, the measurement parameters can be kept constant within 30-50%
(as exemplified by the microarray data reported by Garcia-Martinez et al. 2004).
When signal S varies more than 30-50% under such experimental conditions, then
(and only then) the signal variations observed could be attributed to biological
changes occurring in the cell under investigation.
Interpreting the results of the measurement of intracellular RNA levels using
the DNA microarray technique even under ideal settings is not simple because it
involves at least four biological steps, i.e., Steps 1, 2, 7, and 8, in Fig. 12.5 . (See also
Fig. 12.22 .) It is generally safe to assume that there exists a 1-to-1 correlation
between S and R C : When S increases, so does R C, and whenever S decreases, so
does R C. But the common error committed by many users of the DNA microarray
technique has been to assume that a 1-to-1 correlation exists between R C and the
rate of Step 1, namely, the transcription (or gene expression) step. Such an inter-
pretation of the R C level is invalid because R C levels are determined not by Step 1
alone but also by Step 2 or the transcript degradation (Ji et al. 2009a).
Since the mid-1990s when the era of the DNA microarray technology began, cell
biologists have been interpreting changes in the RNA levels measured with
microarrays almost invariably in terms of transcriptional activation, or more
generally called “expression,” of corresponding genes, i.e., increased rate of
Step 1 in Fig. 12.5 (Alon et al. 1999; Troester et al. 2004; Rhodes and Chinnaiyan
2004; Tu et al. 2005), ignoring transcript degradation, i.e., Step 2 in Fig. 12.5 .This
is surprising since it has been known for a long time that RNA molecules are
unstable toward degradation (Shapiro et al. 1986; Hargrove and Schmidt 1989;
Wang et al. 2002; Yang et al. 2003).
The direct experimental evidence for the critical role that the transcript degradation
step plays in determining transcript level (TL) came to light when two groups - Fan