Isolation of Total and Poly A+ RNA from Animal Cells
Most RNA in a mammalian cell consists of 28S, 18S, and 5S ribosomal RNAs together with tRNAs and other small ubiquitous RNAs. The remainder (<5%) consists of messenger RNA encoding most of the polypeptides of interest to the vast majority of present day biologists. This mRNA is heterogeneous in size and generally carries long tracts of polyadenylic acid (polyA) at its 3′ end. The mRNA can be purified away from other nucleic acids by hybridization of the poly A tract to oligo(dT) as described below (1). Such RNA is referred to as polyA+ RNA and is a superior substrate for many techniques. However, purifying such molecules presents special problems as a result of the inherent instability of some RNAs and the presence of potent RNAse activities in many cell types. Suitable purification protocols should include RNase inhibition or inactivation measures. The tried and tested methods described below include the latter (2,3). Denaturation of all cellular proteins, including RNases, at a rate superior to that of RNA hydrolysis eliminates RNA degradation. This can be achieved using guanidinium thiocyanate and β-mercaptoethanol which denature cellular proteins and disrupt disulfide bonds respectively.
KeywordsCesium Chloride Guanidinium Thiocyanate Polyadenylic Acid Sweet Smell Resuspend Cell Pellet