PolyA+ RNA

Total RNA or (polyA+)RNA (mRNA) can be used for experiments. In the latter case, a purification step is necessary, as mRNA is isolated from total cellular RNA by affinity chromatography on oligo-dT immobilized to a solid support. The amount of the purified RNA is determined by its dual wavelength absorbance at 260 nm and 280 nm and the quality checked by agarose gel or capillary electrophoresis. 

Northern Blot Hybridizations. Total or polyA+RNAs were denatured with dimethyl sulfoxide and glyoxal, size fractionated on 1% agarose gels, and transferred to nitrocellulose filters. Labeled DNA probes were denatured by boiling and hybridized to filters for 16 hr at the appropriate temperature in 50% formamide, 25 mM phosphate buffer pH 6.8, 5X SET (IX SET - 150 mM NaCl, 20 mM Tris pH 7.8, 1 mM EDTA), 0.1% sodium dodecylsulfate (SDS), 10% dextran sulfate,... 

Actin, Tubulin, and 3-Lipoxygenase have Similar Expression Patterns that are Different from those of SSP Genes. Table I summarizes the results of Northern gel blot analysis of polyA+RNA from embryos at 8, 13 and 25 DPF with respect to the expression of actin, tubulin, and B-LOX. The degree of expression of respective transcripts are reported as percent of maximum cpm of probe hybridizing to total RNA. Each of these genes shows the same general expression pattern although actin radioactivity declines more rapidly than that of tubulin, which in turn declines more rapidly than B-LOX. 

Elute the polyA RNA with 2 column vol of elution buffer. 

The eluate will still contain a high proportion of polyA RNA. Adjust the concentration of NaCl in the eluted sample to 0.5Af and repeat steps 4-7. 

Elute the polyA RNA with 2 column vol of elution buffer into a microcentrifuge tube. Add sodium acetate to 0.3Mand 2,5 vol absolute ethanol. Precipitate the RNA at -70°C for 15-30 min. 

Usually, 1-2% of total RNA will be recovered as polyA" RNA by the method described. Many commercial kits utilizing a variety of approaches are now available to achieve the same aim. 

One gram of oligodT should bind 1 mg of polyA+ RNA. It may be regenerated by washing with 0. IM NaOH, sterile distilled water, and column wash buffer. 

Sample preparation RNA should be thawed on ice and an appropriate amount (generally 10-20 pg total RNA or 0.5-1 pg polyA RNA per lane) transferred to a sterile Eppendorf tube. Add 4 vol of RNA sample buffer and place at 65 C for 10 min. Transfer immediately to ice for 2... 

The amount of RNA loaded per dot is obviously dependent on the abundance of the transcript in the RNA population. For a cDNA clone identified by differential screening (and hence of high abundance in the mRNA pool) a loading of 5 ig total RNA per dot should be sufficient. For medium and low abundance transcripts it may be necessary to load more total RNA or isolate polyA RNA for efficient detection of transcript. 

Analv.si.s of mRNA levels and chromosomal location of genes Isolated polyA+ RNA was electrophoresed under denaturing conditions and transferred to a nylon membrane filter. Restricted genomic DNA was fractionated in agarose gels and also transferred to a nylon membrane (2). Probes were labelled with P using a random priming method. 

In this study we isolated chloroplasts of normal and heat shocked Chlamydomonas reinhardtii cells, PolyA-RNA was purified from heat shocked cells. In vitro translation products of this RNA were used to investigate import into chloroplasts derived from normal and heat-shocked cells. 

PolyA RNA from heat shocked Chlamydomonas reinhardtii was isolated as described (8), This RNA was translated in vitro in a reticulocyte lysate. Translation products were identified by indirect immunoprecipitation as described (12). 

Translation of polyA RNA from heat shocked cells showed two major products of 22 and 24 kDa (Figure IB). The products were identified with specific antibodies as 22 kDa heat shock protein and the 24 kDa precursor protein of SS. 

Figure 1 B Uptake of products from in vitro translation of polyA RNA from heat shocked Chlamydomonas in the reticulocyte lysate 9) products of control incubation without added RNA 10) translation products of polyA-RNA from heat shocked Chlamydomonas reinhardtii 11) in vitro translation products of polyA-RNA associated with washed control chloroplasts 12) in vitro translation products associated with chloroplasts isolated from heat shocked cells. 13) products in recovered, trypsin treated control chloroplasts 14) products in trypsin treated heat shock chloroplasts,...

The quality of RNA used in a microarray experiment is probably the single most important determinant of success. We always isolate, and subsequently work with, total RNA in preference to polyA+ RNA. The protocols that follow have been used to extract RNA from embryonic material, adult tissue, and cell lines. It is important to wear gloves and eye protection during the homogenization steps. 

Figure 1. Comparison of different methods for RNA isolation with the use of guanidinium thiocyanate. [ S]methionine-labeled peptides were synthesized from purified polyA+ RNA in a rabbit reticulocyte lysate (18). The polyA+ RNA samples (1 yg per lane from adult rat pancreas) used for translation were from the total RNAs isolated by the following methods Lane 1 ethanol precipitation method (3). Lane 2 LiCl method (17). Lane 3 low temperature method (1). X indicates unidentified low molecular weight polypeptide.

Figure 4. Autoradiogram of [-methionine labeled peptides synthesized in rabbit reticulocyte lysate and immuno-precipitated by a rabbit anti-insulin serum. The RNAs used were 1 pg of Brome Mosaic Virus as a control (lane 1), no RNA (lane 2), 0.5 yg of polyA+ RNA isolated from rat pancreas (lane 3), 0.2 yg of mRNA synthesized by T7 polymerase (lane 4), 0.2 yg of polyA+ RNA combined with 1 yg of antisense mRNA synthesized by SP6 polymerase (lane 5), and 0.2 yg of synthetic mRNA combined with 1 yg of antisense mRNA. The RNAs used in lane 5 and 6 were heated at 70°C for 15 min and cooled slowly before the translation reaction.

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