Poly A mRNA isolation

The quality of mRNA is important in generating recombinant antibodies. It is particularly important to ensure minimal ribosomal RNA contamination when cDNA is random primed for Ubraiy construction to reduce the proportion of clones which represent non-mRNA species. The spleen mRNA of immunized mice is presmnably derived mainly from plasma cells, as the level of Ig mRNA in these cells is up to 1000-fold greater than in resting B cells. For spleens, extraction of total RNA followed ly mRNA isolation on oligo(dT)-cellulose is recommended in order to effectively process the large number of cells present in a spleen. Cytoplasmic RNA Extraction Kit (see Protocol 2) followed by mRNA Purification Kit is hi y recommended. 

Observe special precautitHis to avoid degradation of mRNA by RNases (see Protocol 2, Recommendations). 

3 M sodium acetate pH 5.2 dissolve 408.3 g sodium acetate.3H20 in 800 ml of distilled water, adjust to pH 5.2 with gladal acetic add, adjust the volume to 1 litre with distilled water 

Follow the manufacturer s instructions for preparation of an oligo(dT)-cellulose spun column (Procedure A). 

Perform one round of purification following the manufacturer s instructions (Procedure B Isolation of mRNA). 

After elution, add lO (i,l of glycogen solution (10 mg/ml, QuickPrep mRNA Purification Kit) per 500 ixl of mRNA. 

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Both proteins have been shown to be translated vitro In a reticulocyte lysate system as preinhibitors, 2000-3000 daltons larger than those synthesized and accumulated vivo (11). The preinhibitors may be Important In the compartmentall-zatlon of the Inhibitors as they are stored In the central vacuole, or plant lysosome, of the plant cells (12). We have now studied the time course of the Increase In translatable mRNA In leaves of wounded plants utilizing poly(A) mRNA Isolated at various times following wounding. 

It is possible to apply RNA chromatography in a preparative mode in order to produce a series of RNA fractions which can be used for subsequent research. One of the major drawbacks of poly A mRNA isolation is that some mRNAs are not polyadenylated and therefore will be excluded from any subsequent analysis. The use of RNA chromatography in a preparative mode offers the potential for isolating at least a fraction of those mRNA species that do not co-elute with rRNA. This is clearly an important area for development, since the analysis of cell-specif-... 

Fig. 2. Time course of accumulation of HSP mRNA. One jUg of poly(A) RNA isolated from soybean hypocotyls after different times of incubation at 42.5 °C (hs) or at additional times after transfer back to 28 °C after 4 h at the elevated temperature (recovery), were electrophoresed in formaldehyde agarose gels. Blots of these gels were hybridised with a mixture of four cDNAs encoding small soybean HSPs ranging from 15 to 23 kDa. From Schoffl Key (1982).

BBB-RNA must be isolated for molecular cloning and analysis of BBB-specific transcripts, and several methods for isolating BBB-poly (A+) mRNA in one or more steps have been described [68]). Li et al. recently reported that they obtained yields of 12 pg poly (A+) mRNA from a single bovine cortical shell and 3.2 pg poly (A+) mRNA from the pooled cerebral hemispheres of 21 rats [70]. 

The isolation of Poly (A+) mRNA has also led to the synthesis of complementary DNA for the preparation of BBB-cDNA libraries and the construction of BBB-re-... 

Etiolated pea seedlings were illuminated as indicated. Poly (A) mRNA was isolated, dotted onto nitrocellulose filters and hybridized to P-labelled ELIP cDNA. 

The antiserum raised against the purified enzyme effectively bound ACC synthase of wounded mesocarp, whereas the non-immune serum did not (Fig. 2). The in vitro translation products in a wheat germ extract directed by poly(A) RNA isolated from wounded mesocarp tissue, but not from fresh mesocarp tissue, contained one major 55 kDa polypeptide which was bound by the antiserum (Fig. 3, inset). The translatable mRNA for ACC synthase increased after wounding in proportion to the increase of endogenous enzyme activity (Fig. 3). 

Figure 5. Size analysis of Inhibitors I and 11 specific mRNA from levels of 9- and 18-h singly wounded tomato plants and 18-h doubly wounded plants. Poly(A ) RNA was applied to 15-30% linear sucrose gradients and was spun at 25,000 rpm. Twenty-five fractions were collected, the absorbency was measured, and the mRNA was precipitated by cold ethanol. In vitro translations were performed with each fraction in a rabbit reticulocyte system, and isolation of the preinhibitors with preformed antibody precipitates located the position of the two inhibitors. The gradients were calibrated by centrifugation of tomato leaf polyfA)" RNA on an identical gradient. The locations of translatable mRNAs for Inhibitors I and II were identical with RNA obtained from 9- and 18-h singly wounded or 18-h doubly...

RNA to initiate cDNA synthesis. All cellular mRNA contains multiple repeats of adenine bases (poly-A tails). Therefore the complementary thymine bases (oligo-dT) can be used as a primer that binds to the mRNA template required for the reverse transcriptase to synthesize the cDNA. In the case of pancreatic mRNAs (Figure 4.2), the signihcantly higher mRNA for insulin compared with other proteins allowed success in isolating the insulin-specihc cDNA. Subsequent insertion of cDNA into a bacterial expression vector allowed the production of functional insulin that is now marketed as a successful therapeutic product (Figure 4.2). 

Probes may also consist of DNA copied from mRNA. This is known as cDNA and is also widely used to determine indirectly the sequences of mRNA molecules. Messenger RNA may be isolated from the total cellular RNA by affinity chromatography on bound poly (dT) or poly (U). These materials selectively hold RNA with the poly (A) tails characteristic of most eukaryotic mRNA (see Chapter 28). Another source of mRNA is polyribosomes (polysomes), which are "reading" mRNA and actively making proteins. 

In addition to genomic libraries, cDNA libraries can be prepared from mixed mRNAs. The total RNA of cells is isolated and passed through an affinity column containing oligo(dT) chains. These bind to the 3 -poly(A) tails of the mRNAs, allowing them to be isolated. The mixed mRNAs can then be cloned using a poly(AT)-tailed plasmid vehicle and a reverse transcriptase.183 184... 

Fig. 3. A northern blot of poly(A)+ RNA probed with a psi cDNA clone showed enhanced levels of psi messenger RNA as phosphate starvation became more severe. Poly(A)+ RNA was isolated from cultures at 3, 6 and 9 days after transfer to —Pi or +Pi medium (A. Danon et al., unpublished data in preparation). Equal amounts of RNA (1 pg per treatment) were separated on a denaturing formaldehyde/agarose gel and used for a northern blot. The filter was probed using a psi cDNA clone (identified by —/+ screening using standard methods) as the probe. Enhanced levels of mRNA for this clone are seen as early as 3 days after transfer to —Pi medium although cell growth equivalent to the unstressed control continued until day 8.

A large number of studies (33) support the thesis (59) that the function of GA may be that of a derepressor of gene activity. Giberellic acid has significant effect on the synthesis of all species of RNA s (63, 64), but the formation of < —amylase does not depend on new synthesis of ribosomal and transfer RNA s. Unequivocal proof for GA-induced formation of transcripts was provided by the in vitro synthesis of peptides that are immuno-logically similar to -amylase on poly A+RNA templates that were. isolated from hormone-treated aleurone cells (65, 66, 67). Of particular significance is the finding that detectable levels of -amylase mRNA s were formed within 2 hr of treatment with GA. 

Control of the synthesis of amylase ntRNA s in barley aleurone cells and the synthesis of cellulase mRNAs in pea epicotyl cells are similar in some respects. The control of cellulase activity in pea epicotyl is the only known example of auxin-induced formation of specific mRNA molecules. The formation of cellulase mRNA was demonstrated by the isolation of poly A + RNA s and in vitro synthesis of cellulase (71) using the protein-synthesizing system of wheat germ (72). The formation of cellulase mRNA precedes the increase in cellulase levels by more than 12 hr. Thus, it appears that the increase in rate of synthesis of translatable cellulase mRNA s in the pea epicotyl (71) and that of -amylase mRNA s in barley aleurone cells (65,... 

Figure 7.4. Isolation of poly-A+ RNA by biotin-streptavidin affinity matrix. Poly-A+ RNA is captured as a hybrid between poly-A+ RNA and biotinylated oligo(dT) by streptavidin matrix. Most mRNAs carry poly-A+ stretch at their 3 end, and hence poly A-containing RNA can be enriched substantially by this affinity capture method. Poly-A+ RNA can be eluted from the beads by low salt or water. The eluted RNA can be ethanol precipitated.

Figure 7.3 Regulation of HMG-R expression in male and female /. paraconfusus. A JH Ill-induced HMG-R mRNA levels in females and males following topical application of 5.3 pg JH III. B Northern blot showing the effect of topical JH III application on HMG-R mRNA in males. The doses applied are shown above the image. In both A and B, poly(A)+RNA was isolated 20 h following treatment. The relative levels of HMG-R mRNA (normalized to the actin signal) compared to untreated males is given below each image. Reproduced from Tittiger et al. (1999) with permission.

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