RNA from cells

Isolation of RNA from cells or tissues is usually carried out in the presence of the chaotropic agent guanidinium thiocyanate with the purpose of disrupting the cellular structures with concomitant inhibition of ribonucleases. In this section, two basic procedures that use guanidinium thiocyanate are presented. The first is a modified version of the original procedure of Chirgwin et al.1 that is appro- 

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There are two sources of RNA that can be used as a reference sample (the green-labeled sample) RNA from cells that are not related to the experiment or unfractionated RNA from the same cells under the same treatment. It is preferable to take an unrelated RNA sample that will be used in all future experiments, thereby serving as a common reference for all conditions and treatments. The most important parameter for this sample is that it yield a strong and reliable signal for as many genes as possible on... 

Fast and simple methods for extraction of DNA or RNA from cells, whole blood, and other fluids have been reviewed by Bloch24 and Kawasaki.25 In general, target DNA is released from the cells or virus by proteinase K digestion in the presence of detergent followed by heat inactivation of the proteinase K. A small aliquot of the cell lysate is then used directly as the template in the amplification reaction. For cell culture supernatant fractions, the proviral DNA released from the lysed cells can be directly... 

Columns of mercuriated cellulose have been applied to the separation of thiol-containing RNA from cells treated with 6-thioguanosine or 4-thiouri-dine. Mercuriated cellulose was prepared by treating cellulose with allyl glycidyl ester followed by mercuric acetate. Prior to modification RNA was purified on oligo(dT)-cellulose columns. 

Yield of total RNA from cells in culture may vary greatly depending on cell type and growth conditions. Typically, one may expect 50-500 ug total RNA from lO" cells. 

Fig. 5. Analysis of RNA complementary to MMTV DNA in 341 cells treated with (ADP-ribose) synthetase inhibitors. Total RNA from cells treated with 10 tM 3-ABm for 0 lanes a, f), 4 (lane b), 16 (lanes c, h), 32 (lane d), and 64 h (lane e), or with 10 mM 3-aminobenzoic acid for 16 h (lane g), was subjected to electrophoresis on 1.2% agarose gels, transferred to a nitrocellulose paper, and hybridized with nick-translated P]-labeled MMTV DNA specific for the env region. The size of the RNA were extrapolated from the migration of radiolabeled Hind III DNA standards (lane i) (4.4, 2.3, and 2.0) kilobase pair (Kbp) and 18S and 28S rRNA from the ethidium bromide staining pattern...

Total or poly (A)+ RNA from cells or tissues of interest (see Note 4)... 

Manipulations of RNA samples should be done with gloved hands. Extract total RNA from cell monolayers by the method of Chomczynski and Sacchi (17) or an equivalent method. 

Soderlund, H., Pettersson, U., Vennstromm, B., Philipson, L., and Mathews, M. B., 1976, A new species of virus-coded, low molecular weight RNA from cells infected with adenovirus type 2, Cell 7 585. 

Wengler, G., Beato, M., and Wengler, G., 1979, In vitro translation of 42 S virus-specific RNA from cells infected with the flavivirus West Nile virus. Virology 96 516. 

The plaque assay is desirable because it is very sensitive and only detects infectious viral particles. However, there are viral agents which cannot be supported by cell lines. In these cases other methods must be used. The polymerase chain reaction (PGR), which amplifies DNA or RNA from viral agents, can be used to detect the presence and quantity of viral agents. The amount of RNA or DNA target in the initial sample can be determined by competitive PGR where the quantity of amplified product is compared to a control PGR product where the initial amount of target is known. Quantification is also possible by an end-point dilution method similar to that used to determine a tissue culture infections dose. PGR methods can be very sensitive however. 

Because different cell types in eukaryotic organisms express selected subsets of genes, RNA preparations from cells or tissues in which genes of interest are selectively transcribed are enriched for the desired mRNAs. cDNA... 

Differential display is a method for identifying differentially expressed genes, using anchored oligo-dT, random oligonucleotide primers and polymerase chain reaction on reverse-transcribed RNA from different cell populations. The amplified complementary DNAs are displayed and comparisons are drawn between the different cell populations. 

RNA oncogenic vimses have an unusual enzyme, reverse transcriptase, which is capable of making DNA copies from an RNA template. Cells transformed by these retrovimses have been shown to possess DNA transcripts of the viral RNA. It appears that the transformahon from normal to malignant is associated with the acquisition by the cell of viral DNA. 

Nasmyth You need more polymerase in a big cell to produce more RNA from the same number of genes. 

GeneReleaser set of proprietary polymeric materials, which facilitates the DNA release from cells or other genetic materials, suitable for PCR amplification leading to a PCR ready DNA/RNA protocol made in about 5 min. 

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