Figure 2.

Comparison of the one-step FAE and hot phenol extraction methods. (A) Outline of a small-scale RNA isolation procedure using FAE extraction. For routine RNA analysis from yeast cultures, a convenient microcentrifuge tube format illustrated here is to start with 1 OD₆₀₀ unit of cells and perform lysis in 30–40 μL of FAE. The typical yield we obtain is ∼22–30 μg total RNA when using mid-log phase BY4741 cells. (B) Spectrophotometric measurements of the yield and purity of RNA isolated from a mid-log phase culture. FAE-extracted sample was analyzed without additional purification. (C) Gel analysis of 3.5 μg RNA prepared using hot phenol (lane 1), one-step FAE extraction (lane 2), and FAE extraction followed by additional purification with RNazol RT (lane 3). Individual RNA species were detected by northern hybridizations. (D) Recovery of different RNAs using FAE and hot phenol extraction. Total RNA was extracted by the 2 methods from the same log-phase BY4741 culture, separated by gel electrophoresis, and transferred to a membrane. The membrane was stained with methylene blue and hybridized with radiolabeled probes to detect the indicated mRNAs, mitochondrial rRNAs, and tRNAs. (E) Large rRNA precursors can be efficiently extracted with FAE. RNA was isolated from cells in log-phase at 30°C or cells that were heat shocked at 39°C for 15 min to suppress productive ribosome biogenesis.¹² 2 μg total RNA (as estimated by A₂₆₀ absorbance) was loaded in each lane. The membrane was hybridized with oligonucleotide probe 006¹² to detect pre-rRNAs.