Nonspecifically bound probe is washed from the membrane (Memelink et al. 1994;
Sambrook and Russell 2001), and the membrane is exposed to X-ray film in autoradio-
graphy (Reece 2004; Sambrook and Russell 2001) or a phosphor screen (Johnston et al.
1990) to develop the image (Fig. 11.9).
There are numerous ways to increase signal on an RNA gel blot. If the RNA is degraded
during extraction, the signal will be weak. Temperatures should be kept low (, 48 C) at most
steps of RNA extractions. RNA gel blot sensitivity can be increased by enriching for
mRNA (Memelink et al. 1994; Sambrook and Russell 2001), and this also decreases the
problematic nonspecific binding of probe to the ribosomal bands. There are in vitro tran-
scription kits widely available for the production of single-stranded RNA probes that can
be radiolabeled to high specific activity for increased sensitivity. Loading more RNA
will also increase the sensitivity of the procedure. Typically, a “housekeeping gene” such
as actin or ubiquitin is also used as a probe along with the GOI to control for differential
loading of RNA among lanes. In addition, or alternatively, the total mRNA can be quanti-
fied on the gel prior to blotting as a loading control procedure for subsequent quantitation of
specific transcript (Fig. 11.9).
11.4.3.2. Quantitative Real-Time Reverse Transcriptase (RT)-PCR. The power
of quantitative real-time PCR has been coupled with the reverse transcriptase (RT) reaction
as a tool to monitor and quantify gene expression. In this procedure, total RNA or mRNA is
extracted and used as a template with a complementary primer, 2^0 -deoxynucleoside
50 -triphosphates (dNTPs), and reverse transcriptase to generate single-stranded complemen-
tary DNA (cDNA) (Reece 2004). The cDNA is replicated in normal PCR using Taq DNA
polymerase. Repetitive cycles of quantitative real-time PCR amplify the sequence of inter-
est, while a fluorescent dye monitors the accumulation of PCR product (Reece 2004). This
method is sensitive and high-throughput and does not require gel fractionation. It does not
require a lot of starting material, but the sequence of the GOI is required for primer design.
Figure 11.9.Northern blot: (a) specific cDNA from a arsenic repressor element is hybridized to
transgenic plant mRNA, indicating that certain transgenic events highly express the transgene; (b)
contains stained total RNA bands indicating approximately equal loading. (Figure courtesy of
Jason Abercrombie.)
11.4. DEFINITIVE MOLECULAR CHARACTERIZATION 285