Biology Reference
In-Depth Information
further calculation steps are required if not only the mRNA amount but also the
amount of total RNA per cell changes between the biological conditions compared.
3
PERFORMING THE EXPERIMENTS
3.1
RNA preparation and quality assessment
The preparation of high-quality RNA is an extremely important prerequisite for
generating reliable transcriptome data, even more so for bacteria, where mRNA
half-lives are short (mostly in the range of a few minutes). The different methods
employed usually follow one of three general techniques: (i) organic extraction using
acid phenol or commercially available solutions of guanidine isothiocyanate and
phenol (TRIzol, TriFast or TRI Reagent) based on the single-step RNA isolation
method developed by
Chomczynski and Sacchi (1987)
, (ii) adsorption of RNA onto
a spin column membrane (silica matrix or glass fibre filter) of commercially avail-
able kits (NB. For isolating total RNA including small RNAs
200 nt spin columns
need to be combined with organic extraction, except for Norgen's Total RNA Puri-
fication Kit.) and (iii) magnetic particle-based (automated) methods. In each case,
the first issue to be considered is maintaining RNA integrity prior to RNA isolation,
that is, during and after harvesting the bacteria. An appropriate procedure involves
centrifugation at low temperature, freezing the cell pellets in liquid nitrogen and
storing them at
<
80
C until cell disruption. To prevent changes in the transcriptome
due to stress responses induced during centrifugation, sodium azide is added to the
harvesting buffer (see
Winter
et al.
, 2011
).
At the beginning of each RNA preparation procedure, bacterial cell lysates are
prepared, usually by enzymatic lysis or mechanical disruption. Efficient cell disrup-
tion while maintaining RNA integrity is particularly an issue for Gram-positive bac-
teria. The method recommended involves mechanical cell disruption followed by
organic extraction with acid phenol (
Eymann
et al.
, 2002
). This method, originally
developed for preparation of high-quality RNA from yeast (
Hauser
et al.
, 1998
), has,
over the past 10 years, been extensively used in studies involving
B. subtilis
,
Bacillus
anthracis
and
S. aureus
. A detailed protocol is provided in
Nicolas
et al.
(2012)
.
Cells frozen in liquid nitrogen are rapidly disrupted by means of a grinding ball
in a Mikro-Dismembrator instrument (Sartorius) followed by completion of lysis
with guanidine thiocyanate as denaturing agent. This procedure effectively protects
the RNA from degradation, yielding RNA preparations with a high degree of integ-
rity as indicated by rRNA precursor bands visible above the 23S rRNA band in gel
electrophoresis (
Figure 6.1
). In general, it is important that the RNA preparation
method results in the complete size range of RNA (not selectively excluding small
or large transcripts) and that the same method is used for all samples to be compared
because different methods are likely to have an impact on the transcriptome.
Before the RNA samples can be used for transcriptome analysis, residual DNA
has to be removed by DNase treatment using, for example, the RNase-Free DNase
