Biomedical Engineering Reference
In-Depth Information
LCM Normal Colon
10
1
0.1
A1
A2
A3
Control
Sample
Figure 6.2
RNA integrity assessment from RNA obtained without extraction. Frozen samples of
four colonic biopsies were catapulted into the caps of microfuge tubes, following LCM using a
PALM LCM system. Three (A1-A3) were processed using Invitrogen's CellsDirect two-step system,
with the RNA subjected to RT-qPCR without a separate extraction step. RNA from the fourth
sample (control) was extracted and purified using Qiagen's RNeasy Mini Kit. RT-qPCR assays were
carried out as described, with separate primers and probes (see Protocol 6.1) quantitating the 5
and 3
ends of the GAPDH mRNA [11]. It is apparent that there are no significant differences in
the 3
:5
ratios between the four samples. This suggests that a separate RNA extraction step is
not required for preserving maximum RNA quality when processing frozen LCM samples.
(see Protocol 6.1). The data obtained are independent of ribosomal RNA (rRNA) integrity,
provide a quantifiable measure of the degradation of the transcripts of interest and are mod-
eled on the standard approach adopted by microarray users and long-accepted conventional
techniques applied to end-point PCR assays [50]. This is in contrast to RNA quality assays
performed using an RNA chip on the Agilent 2100 Bioanalyser or BioRad Experion plat-
forms that provide a measure of rRNA integrity, which may or may not reflect in the quality
of the (largely unmeasured) mRNA. The 3
:5
assay described in Protocol 6.1 measures the
integrity of the ubiquitously expressed mRNA specified by the GAPDH gene, which is taken
as representative of the integrity of all mRNAs in a given RNA sample. However, since
different mRNAs degrade at different rates, this may not always be the case and it may be
necessary to design similar assays for specific targets. The RT reaction of the 1.3 kb GAPDH
mRNA is primed using oligo-dT, and a separate multiplex PCR assay is used to quantitate
the levels of three target amplicons. These are spatially separated with one towards the 5
end, the second towards the center and the third towards the 3
end of the mRNA sequence.
The ratio of amplicons reflects the relative success of the oligo-dT primed RT to proceed
along the entire length of the transcript. Clearly, the progress of the RT enzyme past the
5
amplicon is dependent on the intactness of the mRNA, with the enzyme unable to reach
it if the mRNA is degraded. Consequently, all things being equal, a 3
:5
ratio of around
