qRT-PCR and sequencing analysis of adenovirus DNA in human sarcomas.

<p>All PCR positive sarcoma specimens are shown. In samples 32 and 42 the laboratory strain with a deletion in the E3 region was detected. In sample 62 a sequence identical for type 2 and type 6 has been identified. GIST: gastro intestinal stroma tumors.</p

HAdV copy numbers in human sarcomas detected by qRT-PCR.

<p>qRT-PCR was performed for the hexon gene of HAdV and for beta-2-microglobulin, B2M (a single human house-keeping gene). The calculation of the viral copies is related to the copy number of the house-keeping gene for each specimen. The virus copy numbers per 10³ cells are shown for 4 leiomyosarcoma (Lei), 7 liposarcoma (Lip) and one GIST (gastro intestinal stroma tumor).</p

Typing of adenovirus strains present in human sarcoma specimens.

<p>E1A has been amplified by nested PCR in species C positive samples. A) A detailed scheme shows the binding sites of the oligonucleotides for the first and second round of the nested PCR. B) 531bps of the E1A gene have been sequenced from 7 different samples. A part of the sequences investigated are depicted in an alignment with two reference GenBank sequences of HAdV-5 (NCBI acc. no.: AY339865) and HAdV-6 (NCBI acc. no.: HQ413315.1).</p

Detection of HAdV by Fluorescence-in-situ-hybridization (FISH).

<p>The hybridization was performed in all experiments with a biotinylated adenovirus DNA probe. Control hybridization was performed on (A) A549 cells infected with HAdV type 5 (24 h, MOI 50) and (B) HEK293. Paraffin-embedded human sarcoma tissue sections were additionally hybridized for the internal positive control (DIG labelled centromeric probe q12). FISH results from (C) a PCR positive leiomyosarcoma (sample 51), (D) a liposarcoma (sample 70) and (E) a PCR negative liposarcoma (sample 47) are shown. For the detection of HAdV-DNA the mouse anti-Biotin-Cy3 antibody (Jackson ImmunoResearch) and for the centromeric probe the sheep-anti-digoxigenin-FITC labelled antibody (Roche) have been used. The bar in picture E represents 10 µm.</p

Oligonucleotides for quantification and detection of HAdV.

a<p>The probe sequences indicated are TagMan probes labeled with 6-carboxyfluorescein on the 5′ end and with 6′-corboxytetramethylrhodamine on the 3′ end; ^b AdVC E1 for n and AdVC E1 rev n are primers for the second round of the nested PCR.</p