A rapid microprocedure for isolating RNA from multiple samples of human and rat brain.
In order to establish a routine procedure for isolating undegraded RNA from small amounts of rat and human brain tissue, several techniques were investigated. Initial studies demonstrated that undegraded RNA could not be reproducibly isolated from milligram amounts of brain tissue homogenized in an aqueous medium. Several isolation techniques utilizing tissue homogenization in the denaturing agent guanidinium chloride were compared. This method of homogenization, followed by sedimentation of RNA through cesium chloride, resulted in good yields of undegraded translationally active RNA. A maximum of 6 RNA samples could be processed simultaneously. In contrast, when homogenization in guanidinium chloride was followed by repeated guanidinium chloride-ethanol precipitations many samples could be processed simultaneously. The resulting RNA yields were low. The introduction of several modifications in the guanidinium chloride-ethanol precipitation technique resulted in a high yield of undegraded translationally active RNA. DNA was removed by two guanidinium-ethanol precipitations. Residual protein was digested with proteinase K. RNA was precipitated after extraction with phenol-chloroform-isoamyl alcohol. This refined procedure allows the recovery, in high yields, of translationally active undegraded RNA which is both DNA and protein free. Thirty-six samples can be processed in one day.
Ilaria
R
R
Wines
D
D
Pardue
S
S
Jamison
S
S
Ojeda
S R
SR
Snider
J
J
Morrison
M R
MR
D000818:Animals; D001923:Brain Chemistry; D002531:Cerebellum / Q000032:analysis; D002540:Cerebral Cortex / Q000032:analysis; D004587:Electrophoresis, Agar Gel; D006801:Humans; D008832:Microchemistry; D012313:RNA / Q000302:isolation & purification*; D051381:Rats
D012313:RNA