is left behind, it will interfere with downstream processes (such
as RNA isolation and gDNA digestion of the RNA sample).
- To prepare the RLT (+βME) mixture, add a 1:100 dilution of
βME (10μlβME per 1 ml RLT). If you do not use all of the
RLT +βME mixture, it can be stored at room temperature for
up to 1 month. Additionally, the pellet at this stage may not be
visible, yet it is important to thoroughly resuspend all the
material.
- Prior to RNA isolation, place the appropriate amount of silica
beads into the bottom portion of a screw cap microfuge tube. I
recommend filling the tube ¼ of the way full with beads. In a
separate autoclavable container, place the corresponding lid.
This can be done in bulk ahead of time by using an autoclavable
microfuge rack to hold the open screw cap microfuge tubes
containing the beads, and then covering the open tubes/rack
with foil. Autoclave the tubes/rack and the container with the
lids. After autoclaving, wear gloves and carefully screw each
sterile lid on each sterile tube containing beads. They are now
ready for future use.
- Depending on your organism, the extent of mechanical disrup-
tion required to lyse the cells may vary. It is recommended to
optimize this procedure by determining the number of repeti-
tions of bead-beating required for lysis. Initially, it is a good
idea to bead-beat similar samples (from same strain, growth
condition, and number of cells) for increasing amounts of
repetitions and then carry each sample through RNA isolation.
At the end, compare the samples for RNA yield and quality to
determine which amount of bead-beating results in the best
yield of undegraded RNA. Mechanical disruption can cause the
samples to heat up. Thus, please ensure that you properly chill
each sample on wet ice if you increase the amount of bead-
beating. Also note that other methods of cell lysis are available,
such as enzymatic disruption. Whichever lysis method is used,
please take care to optimize it for each organism and sampling
time (as sometimes stationary phase samples are harder to lyse).
Note that enzymatic lysis often requires extended incubation
periods at 37C; this is not optimal for RNA isolation as RNA
molecules have short half-lives and may degrade. Thus,
mechanical disruption is recommended, if available.
- If there is gDNA contamination in your RNA sample, you will
observe a PCR product. Even a faint band means that there is
gDNA contamination, and the sample cannot be used. If this
occurs, alter the conditions for the TURBO DNase Digestion
(seeSubheading3.1,step9) by using half the amount of
starting RNA material. Never repeat digest the same RNA.
RNAseq of QS Regulons 189
