Biomedical Engineering Reference
In-Depth Information
resource for the elucidation of disease mechanisms and validation of differentially expressed
genes as novel therapeutic targets or prognostic indicators.
RNA extracted from FFPE material is a little more difficult to analyze [51], as exten-
sive degradation can occur before [52] or during [53] the formalin fixation process. Fur-
thermore, formalin fixation generates cross-links between nucleic acids and proteins and
covalently modifies RNA, making subsequent RNA extraction, RT and quantification anal-
ysis problematic [54]. Not surprisingly, fixatives are important [55] and different tissue
preparation methodologies will invariably lead to different results from different labora-
tories. However, since real-time RT-PCR amplification generates amplicons that are as
small as 60 bp, this technique is suitable for estimating mRNA levels from such tissue
samples with best results when a specific gene reverse transcription primer is used [56-60].
Various methods can be used to assess the presence of inhibitors within biological sam-
ples. The PCR efficiency in a test sample can be assessed by serial dilution of the sample
[61], although this is impossible when using very small amounts of RNA extracted, for
example, from single cells or from laser capture microdissected sections. Alternatively,
there are mathematical algorithms that provide a measure of PCR efficiency from analy-
sis of the amplification response curves [62-64]. Internal amplification controls (IACs) that
co-purify and co-amplify with the target nucleic acid can detect inhibitors as well as indicate
template loss during processing [65]. Another approach utilizes a whole bacterial genome
to detect inhibition from clinical samples [66]. A recently described qPCR reference assay
identifies inhibitors of the RT or PCR steps by recording the
C
t
values characteristic of
a defined number of copies of an artificial sense-strand amplicon [15] (see Protocol 6.2).
Inhibitors in the RNA sample will result in an increased
C
t
value when the amplicon is run
in the presence or absence of RNA samples.
PROTOCOL 6.2 Detection of RT-qPCR Inhibitors
Equipment and reagents
RNA template (50-500 ng/ul) or cDNA (1 : 10 dilution of cDNA synthesis)
g
•
•
2
×
commercial qPCR master mix buffer (containing dNTPs and chemically or antibody
inactivated thermostable polymerase) without MgCl
2
•
25m
M
MgCl
2
SPUD synthetic DNA amplicon (5
-3
):
AACTTGGCTTTAATGGACCTCCAATTTTGAGTGTGCACAAGCTA
TGGAACACCACGTAAGACATAAAACGGCCACATATGGTGCCATGTAAGGATGAATGT
(Sigma Genosys; prepare a stock 20
μ
M
solution which contains 1.2
×
10
13
molecules/
μ
l; dilute to an approximately 20 000 copies/
μ
l working solution)
•
SPUD F and R primers (10
μ
M
each) (5
-3
):
forward primer: AACTTGGCTTTAATGGACCTCCA
reverse primer: ACATTCATCCTTACATGGCACCA
•
