11.4.2 Determining the Presence of Intact Transgenes or Constructs
DNA gel blots are also used to determinewhethera particular transgene (e.g., promoter: coding
region:terminator) has been transferred in its entirety, using an enzyme digest that cuts on either
side of the transgene (see Fig. 11.6,HindIII digest). This type of analysis will augment
copy number but is not proof of transformation on its own (Birch 1997; Potrykus 1991).
11.4.3 Transgene Expression: Transcription
11.4.3.1. Northern Blot Analysis.RNA gel blots or northern blots (Thomas 1980)
are used in hybridizations with complementary probes to detect, quantify, and size tran-
scripts and monitor tissue-specific transgene expression at the mRNA level (Figs. 11.8
and 11.9). Like Southern blots, northern blots require large amounts of intact nucleic
acids, but unlike Southern blots, no restriction digestion is necessary—individual tran-
scripts are already naturally size-fractionated. Unlike DNA, RNA is rather unstable; for
instance, it can be degraded by enzymes that naturally chew up RNA—RNases—the
curse of all sorts of RNA analysis for researchers. RNase contamination of samples can
occur easily; the ubiquitous RNase enzymes are present on hands, plasticware, glassware,
and in distilled water. All solutions, glassware, tubes, and tips should be RNase-free to
prevent degradation (Sambrook and Russell 2001). In RNA gel blots, total RNA or
mRNA is separated according to size in agarose gel electrophoresis. RNA also has a nega-
tive charge and behaves like DNA in gel electrophoresis. There is a tendency; however, for
RNA to form secondary structures (it can fold back on itself from hydrogen bonding
between complementary bases); therefore, formaldehyde or glyoxal are denaturants
added to the gel and sample buffer to eliminate intermolecular interactions (Memelink
et al. 1994; Sambrook and Russell 2001). If formaldehyde is used, it must be washed
from the gel before the RNA will transfer to the membrane. The gel is blotted to a nylon
membrane to which the RNA migrates through capillary action. The transcript is detected
by hybridization of the membrane with a complementary radiolabeled probe (Feinberg and
Vogelstein 1983, 1984) or nonisotopically labeled probe (Langer et al. 1981; Memelink
et al. 1994; Sambrook and Russell 2001); the probe may be single-stranded DNA or
single-stranded RNA (Memelink et al. 1994; Sambrook and Russell 2001).
Figure 11.8.The process of RNA gel blot analysis (northern blotting and hybridization): (a) single-
stranded RNA fragments are separated according to size on an agarose gel; (b) RNA fragments are
transferred to a blot; (c) autoradiograph of the blot after hybridization with radiolabeled complemen-
tary single-stranded DNA or RNA probe.
284 TRANSGENIC PLANT ANALYSIS