533558814
HERV-W related RNA detected in plasma from individuals with recent onset
schizophrenia or schizoaffective disorder
Håkan Karlsson1*, Johannes Schröder2, Silke Bachmann2, Christina Bottmer2, Robert H
Yolken3
Methods
RNA was isolated from FLK-cell supernatants containing live bovine leukemia virus (BLV,
kindly provided by Dr. Malik Merza, Svanova Biotech AB, Uppsala, Sweden) and from citrateplasma (Cell Preparation Tubes, B-D Preanalytical Solutions, Franklin Lakes, NJ, USA) from an
individual with no history of mental disease, spiked with serial, 10-fold dilutions of the BLVcontaining supernatant.
Following 0.45 μm filtration (Ultrafree-MC, Millipore, Bedford, MA, USA), RNA was isolated
from 0.15 ml of the flow-through by use of the Viral RNA isolation kit from Qiagen GmbH
(Hilden, Germany) according to the manufacturers instructions.
Contaminating DNA was removed by the use of DNase I (Invitrogen, Groningen, The
Netherlands). Complementary DNA was generated from 10 l of DNase-treated RNA using the
SMART PCR cDNA Synthesis Kit (Clontech, Palo Alto, CA, USA) according to the
manufacturer’s instructions.
PCR was performed in 25 μl reactions using the following reagents; 1x Advantage 2 cDNA
polymerase mix (Clontech), 1x PCR Buffer mix (Clontech) with the addition of 200 μmol/l of
dNTPs (Invitrogen), 1 μmol/l of gene specific primers, see table I, and 1 μl of cDNA template.
All PCR reactions were performed with the following cycling conditions in a GeneAmp PCR
System 9700 (Applied Biosystems, Foster City, CA, USA); heat activation for 2 minutes at 94˚ C
followed by 45 cycles of 94˚ C for 30 sec and 68˚ C for 1 min., with a final extension for 7
minutes at 72˚ C. PCR products were electrophoresed in an agarose gel, stained in 1x SYBR
Gold (Molecular Probes, Leiden, The Netherlands) and visualized on a Gel Doc 2000 gel
documentation system (Bio-Rad, Hercules, CA, USA). PCR products were ligated into plasmid
vectors (pCR 2.1-TOPO, Invitrogen) and sequenced.
Table I. Primer sequences, product size and accession numbers of the GenBank sequences
used for primer design. *size of product wo/w intron.
Target
Orientation Sequence (5’→3’)
Product Accession
size
no.
HERV-W Sense
CCAAGCAGTGAGAGGAGGAG
241 bp
AF156961
gag
Anti-sense
AAACTCTCGGGCTGCAGTTA
GAPDH
BLV
Sense
Anti-sense
Sense
Anti-sense
CGACCACTTTGTCAAGCTCA
TTACTCCTTGGAGGCCATGT
CCCTACAACCCCACAAGTTCGG
ATGGTGTAGCTCCCATCTGGTCTT
97/201*
bp
183 bp
J04038
K02120
533558814
M
-
1 2 3 4 5 6 7 8 -
M
Gel image of 183 bp RT-PCR-products generated by the BLV-specific primers on RNA extracted
from cell culture supernatant, lane 1, and supernatant diluted 10, 100 and 1000 times in human
serum (lanes 2-4), human serum with no BLV-particles added, lane 5. Negative control for the
extraction procedure, lane 6, 1st-strand cDNA synthesis, lane 7, and 19 cycle SMART-PCR step,
lane 8. Negative controls for the BLV specific PCR are indicated by (-). M indicates size marker.