APPLICATION NOTE #201503

RNA Extraction from Sacchromyces cervisiae on the Bead Mill 24
Shari Garrett

Via fermentation, Saccharomyces cerevisiae
has been used for thousands of years forbaking and in the production of alcoholic
beverages. Particularly it is one of the firstorganisms to have its genome sequenced.
As a result, yeasts like S. cerevisiae
are one of the easiest single-celledorganisms to study due to their short
generation time and feasibility to culture.
Also, many of their essential cellular
processes are similar to those found in
humans making it an excellent modelorganism for understanding biochemical,
cellular and molecular interactions foreukaryotic organisms. Traditionally,
yeast RNA and intracellular proteinsare extracted from cells by enzymaticmethods. These enzymatic methods
can be time consuming and can lead tothe denaturation of some intracellularproteins. Mechanical disruption is often
needed to effectively lyse the cells andrelease biological molecules. Beadmill homogenizers, such as the Bead
Ruptor 24, can quickly and effectively
disrupt yeast cell walls for extractionof compounds such as RNA. Herein, weevaluate the potential for extraction of
RNA from S. cerevisiae cells on the Bead
Ruptor 24 bead mill homogenizer. The
extraction efficiency and analyte integrity
was evaluated.

Materials & Methods
Equipment•

Qiagen RNeasy Mini Kit (Cat #74104)•Bead Mill 24 (Cat No. 15340163)

RNA Extraction and Separation
A 40 ml nutrient broth culture of
S. cerevisiae was grown for 3 days at 30°C.
After incubation, the cell density at OD
600 nm was determined to be 0.013
resulting in 5.07 x 106 cells in the originalculture. The cells were harvested bycentrifugation at 12,000 rpm at 4°C for 5minutes. The supernatant was removed
and resuspended in lysis buffer with2-mercaptoethanol (Bio-Rad #161-0710)
provided from the RNeasy kit. The cellsuspension was added to a 2 ml tube
containing 0.5 mm glass beads andprocessed on the Bead Mill 24 for two 30
second cycles at 6 m/s with a 45 second
dwell. The beads were allowed to settle
and 300 µl of the lysate was removed andadded to a clean 1.5 ml microcentrifuge
tube. RNA extraction was carried out per
the manufacturer’s instructions using theQiagen RNeasy Mini Kit. 1 µl of the 40 µl
RNA eluant was quantified on a NanoDropSpectrophotometer (Thermo Fisher) todetermine RNA yields.
1 µl, 2 µl and 4 µl of the eluant was mixedwith 5 µl of TBE/Urea sample buffer(Bio-Rad Cat# 161-0768) and heated at70°C for 4 minutes. The samples werediluted to 10 µl with DD H20. The RNA wasseparated on 1.2% TBE agarose gel at 60 Vand stained with ethidium bromide (BioRad Cat#161-0433) for 30 minutes. The gel
was washed with DD H20 for 10 minutesand visualized on a GelDoc EZSystem(Bio-Rad).

Results
Yeast cells have an extremely robust
cell wall comprised of cross-linking

polysaccharides and proteins. Lysing thecell wall is the primary obstacle whenattempting to recover nucleic acids. In thisapplication, we evaluated the capability of
the Bead Mill 24 to disrupt yeast cells forthe purpose of extracting high quality RNA
from S. cerevisiae. The extractionof RNA from S. cerevisiae is critical as it
is a common model organism used asa scientific tool in understanding thecellular and biochemical interactions ofmany organisms.

Figure 1 RNA Agarose Gel Electrophoresisof S. cerevisiae lysate: Lane 1. New England
Biolab ssRNA Ladder. Lane 2: 1 µl extracted
RNA. Lane 3: 2 µl extracted RNA. Lane 4: 4 µl
extracted RNA
After extraction, RNA yields were
quantified by spectrophotometry. The
RNA yield average was 224.4 ng/µl. The
RNA was then separated and visualized bygel electrophoresis (Figure 1).

Results (cont.)
Based on the gel analysis, the extracted RNA was of good quality with a sharp and clear 26S and 18S rRNA bands. The 28S band is twice as
intense as the 18S band indicating that the RNA is completely intact. The bands also depict the expected size of 2.0kb and 3.8kb for the18S and 26S subunits respectively.

Conclusion
The Bead Mill 24 is capable of lysing S. cerevisiae is less than 5 minutes of processing time for the extraction of RNA in excess of 200 ng/µl concentrations. Gel electrophoresis indicated that the RNA was intact and at the expected molecular weight of the 28S and 18S
ribosomal subunits.

Bead Mill 24: (Cat No. 15340163)