3
sample. The three normalizing genes were selected from a total of six housekeeping genes (ACTB,
B2M, GUSB, PPIA, RPLPO, TUBB). The primers (table below) were designed using Primer3.
7
5’-AGGAGATCTAAGCAGCGTTCC-3’
5’-CCCTCCCATTTAAGGTGGTTA-3’
5’-ACTGGTCTTGACCACGAGCTA-3’
5’-CCCTCCCATTTAAGGTGGTTA-3’
5’-GTCCTGGAATTCCTTGGTAGC-3’
5’-TCATCAGGCAGCTCTTCTAGC-3’
5’-CTCACTCGAGACCACTCCAAG-3’
5’-CTGGAGGATGGTTTTCTGACA-3’
5’-CACAGAGTGGGGCTCAGTTAG-3’
5’-AAACCAGGAAGGAGACGAGAG-3’
5’-AAGAAGACCAGCCTTCCAGAC-3’
5’-TTCGAGGCAGTTTTCACTGAG-3’
5'-TAGAGGTGGGGAGCAGAGAA-3'
5'-TCCCCCAAATTCTAAGCAGA-3'
5'-TGGTGTTTGGCAAAGTGAAA-3'
5'-TCGAGTTGTCCACAGTCAGC-3'
5'-TCATCAACGGGTACAAACGA-3'
5'-GCCTTGACCTTTTCAGCAAG-3'
miRNA expression detection
Total RNA (20 ng) was taken for cDNA synthesis in volume of 20 μl using miRCURY LNA
TM
Universal
RT kit (Cat #203300, Exiqon, Vedbaek, Denmark ). After synthesis the cDNAs were diluted 80-fold
and 4 μl was taken for QRT-PCR. Specific miRNAs (hsa-miR-127, -136, -410, -432, and -433) were
detected with commercial SYBR Green QRT-PCR assays from Exiqon (Vedbaek, Denmark) on ABI
StepOne Plus apparatus (Applied BioSystems, Foster City, CA, USA). The same three housekeeping
genes (B2M, PPIA, RPLPO) were used as above.
Western blot
Solubilized protein samples (20 – 30 μg) in 4M urea (see Sample Preparation section) were applied
to commercial gradient SDS gels (4 - 15%, Cat#456-1084; BioRad), electrophoresed for 1h at 160V.
Blotting (semidry TransBlot SD apparatus; BioRad) onto 0.45μm nitrocellulose filter paper (Cat# 162-
0115, BioRad)was performed at 25Vfor 45 min essentially as described.
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Blots were blocked in 2.5%
skimmed milk. Primary anti-RTL1 (rabbit polyclonal diluted 1:100, Cat# AB107990) was purchased
from Abcam (Cambridge, United Kingdom) and used as suggested by the supplier. Goat anti-rabbit
secondary antibody used in Western blotting was purchased from Thermo Scientific (Pierce
Cat#31460; Thermo Scientific, Waltham, Massachusetts, USA). Blots were developed using Pierce
ECL2 Western Blot substrate (Cat#32132, Thermo Scientific, Waltham, Massachusetts, USA) and
digitized using Typhoon 9410 Molecular Imager (Amersham; Buckinghamshire, United Kingdom).
Immunohistochemistry and immunofluorescence
Paraffin-embedded human atherosclerotic lesions and mammary arteries were cut into thin (4 µm
thick) sections, deparaffinized in xylene (2 x 10 min), rehydrated in graded alcohol series (100%,
95%, 70%, 50%, 5 min each), washed once with PBS (5 min) followed by 0.25% TritonX100 in PBS (10
min) and endogenous peroxidase inactivation (3% H2O2 in PBS, 20 min, done only in case of
immunohistochemical antigen detection). Antigen retrieval was performed by boiling the samples (in
microwave oven using 10 mM TRIS, 1 EDTA, pH9.0, 15 min and followed by cooling down to room
temperature, 30 min). Slides were rinsed in PBS (5 min) and blocked against unspecific binding (2%
human serum, 10% horse or goat serum, 1% BSA in PBS, 30 – 60 min at room temperature). Primary
antibody (diluted in blocking solution) incubation was carried out over night at +4oC. On the next
day, slides were washed with PBS (3 x 5 min) and incubated with secondary antibody in blocking
solution for 30 – 60 min.
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