Centrifugal elutriation of large fragile cells: isolation of RNA from fixed embryonic blastomeres.
In order to analyze the RNA populations present in different cells of very early embryos, we have developed a protocol to purify these large blastomeres using counterflow centrifugal elutriation (CCE). This procedure employs ethanol fixation to stabilize the cells against shear forces encountered during CCE. Using this method, we fractionated the three different blastomere types of the 16-cell sea urchin embryo, the micromeres, mesomeres, and macromeres, achieving 96, 94, and 96% mean purities, respectively. We show here that intact RNA is recovered with equal efficiency from each blastomere preparation. Using this method, we have identified several RNAs that are distributed non-uniformly among these cells.
Nasir
A
A
Department of Biology, University of Rochester, New York 14627.
Reynolds
S D
SD
Keng
P C
PC
Angerer
L M
LM
Angerer
R C
RC
D000818:Animals; D001757:Blastomeres / Q000737:chemistry*; D002469:Cell Separation / Q000379:methods*; D002498:Centrifugation; D000431:Ethanol; D005404:Fixatives; D012313:RNA / Q000302:isolation & purification*; D012617:Sea Urchins
D005404:Fixatives; D000431:Ethanol; D012313:RNA