Isolation of total RNA from spleen

Because we are dealing with a relatively large number of cells (1-3 x 10 splenocytes), we recommend first isolating total RNA from the entire spleen, followed by mRNA isolation (see Protocol 3). 

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. 

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