2. ENVIRONMENTAL ChEMISTRy & TEChNOLOGy 2.1. Lectures

Chem. Listy, 102, s265–s1311 (2008) Environmental Chemistry & Technology
Fig. 2. Sampling area
tant ecological area, which covers 543 km 2 with a great variety
of ecosystems and shelters wildlife including thousands of
European and African migratory birds, fallow deer, Spanish
red deer, wild boar, European badger, Egyptian mongoose,
and endangered species such as the Spanish Imperial Eagle
and Iberian Lynx. This non-contaminated area was used as
Fig. 3. Efficiency curves in genetic correlation between M.
musculus and M. spretus for Gsta3 transcript. ● specific primers,
○ non-specific primers. X-axis, log total mNRA (pg); Y-axis,
threshold cycle (Ct)
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negative reference (SOL) in comparison to the neighbouring
“Domingo Rubio” stream (DR1 to DR6) and the positive
references (PS and ARZ). These areas are contaminated by
mining, agricultural and industrial effluents (DR1 to DR6 and
PS) and by pesticides and fertilizers (ARZ) (Fig. 2.).
The mouse Mus spretus is an aboriginal species that
has been commonly used for environmental assessment
of this area (x) by means of classical biomarkers, but new
tools based on recent –omics, such as transcriptomimcs and
proteomics constitute a promising alternative. However, the
molecular biology methodologies present problems by the
fact of poor inclusion of typical bioindicators in gene/protein
sequences database. For this reason we use species close
to model organisms that are well covered in public databases.
This is the case of M. musculus, which is studied comparatelly
with M. spretus. These comparisons are only possible
if genetic homology between both species is proved.
G e n e t i c H o m o l o g y B e t w e e n M .
m u s c u l u s a n d M . s p r e t u s
A crucial start-point in quantitative RT-PCR is primer
design. For absolute transcript quantification it is necessarily
to design primers that amplify the targets and the calibrator
with optimal (100 %) PCR efficiencies. This fact requires
a great genetic homology between target and calibrant
species. Primers for RT-PCR quantification of M. spretus
Cyp and Gst mRnAs were designed based on known gene
sequences from M. musculus. Remarkably, these primers,
when amplified in M. spretus, gave single products exhibiting
in most cases 100% nucleotide sequence identity. Therefore,
most designed primers were exactly complementary to the
desired M. spretus templates and amplified them with 100%
efficiency. In few cases, however, primers should be redesigned
based on nucleotide sequences of PCR fragments from
M. spretus. Fig. 3 ref. 8 .
Therefore, genetic homology between model and bioindicator
species was clearly proved.
Fig. 4. Sampling area-associated differences in M. spretus hepatic
mRNA levels. y-axis, nRNA molecules/pg total RNA. white
bar, SOL non-polluted area; red bar, polluted area