MRNA isolation

In order to know if the presence of mature proteins correlated with the presence of mRNA, two differents experiments were carried out. In vitro translation of total mRNA isolated from mycelia growing in inducing and noninducing conditions showed similar patterns in both situations except for two bands present only in the former, between 29 and 33 kDa (Fig-4). These bands could correspond to the polypectate lyases. The patterns of both situations showed a common band, more intense in micelia grown in pectin, near 50 kDa wich could correspond to the deglicosilated PG of FORL with a Mr of 50 kDa. 

Figure 5. 1% agarose gel showing PCR amplification products for PG cDNA obtained from mRNA isolate. Lanes 1, 2, 4 and 6 apple pectin culture medium, lanes 3, 5 and 7 glucose culture medium, lanes 1, 2 and 3 to isolate r,3 and lanes 4, 5, 6 and 7 to isolate rz. Lane 8 corresponds to the amplification of the 742 pb probe cloned in the vector PCR TM (In-vitrogen) digested with Eco R1 and lane 9 A digested with Pstl. 

In the last few years, DNA microarrays have been utilized to perform simultaneous analysis of the translational status of thousands of mRNAs, thereby enabling a comprehensive view of translation efficiency and regulation. Analyses were performed on mRNAs isolated from various... 

Cytoplasmic injection of RNA is the most widely used approach. Examples of the types of RNA that are used include mRNA isolated from tissue and cRNA synthesized by in vitro transcription. 

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. 

If you were interested in isolating a cDNA for human serum albumin, why would you use a cDNA library established from mRNA isolated from liver ... 

To obtain a template for the RACE, RNA isolation is necessary to rewrite it into cDNA. Precautions have to be taken, as RNA is a considerably more unstable molecule than DNA and, to add to the situation, its degrading enzymes are among the most stable known. Nowadays, most suppliers provide RNAse-free buffers and water, so DEPC (diethylpyrocarbonate) treatment to destroy RNAse activity is necessary only for home-made buffers. Kits for RNA preparation, either for total RNA or for mRNA isolation, do not require any DEPC treatment... 

Yet another approach for mRNA isolation is the use of oligo(dT)-coated magnetic beads for mRNA isolation (Figure 7.5). Such beads with attached oligo(dT) are available commercially (Dynal, Lake Success, NY). Alternatively, the oligonucleotide can be chemically attached to magnetic beads [29]. 

Jiang, G., Harrison, D.J., mRNA isolation for cDNA library construction on a chip. Micro Total Analysis Systems, Proceedings of the 4th TTAS Symposium, Enschede, Netherlands, May 14-18, 2000, 537-540. 

Cholesterol feeding. Mice were divided into four groups, two of which were fed a normal diet and two of which were fed a cholesterol-rich diet. HMG-CoA reductase mRNA and protein from liver were then isolated and quantified. Graph A shows the results of the mRNA isolation. 

Step 2 Gene isolation (three alternative methods) Nucleic acid triplet sequencing for amino acid sequence mRNA isolation, with reverse transcriptase and complementary DNA DNA probes from genetic library... 

RNase protection assay. This is a sensitive method to determine (1) the amount of a specific mRNA present in a complex mixture of mRNA and/ or (2) the sizes of exons which comprise the mRNA of interest. A radioactive DNA or RNA probe (in excess) is allowed to hybridize with a sample of mRNA (for example, total mRNA isolated from tissue), after which the mixture is digested with... 

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