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SV40 associated miRNAs are not detectable in mesotheliomas.

Gee GV, Stanifer ML, Christensen BC, Atwood WJ, Ugolini D, Bonassi S, Resnick MB, Nelson HH, Marsit CJ, Kelsey KT - Br. J. Cancer (2010)

Bottom Line: The Institute of Medicine has recommended the development of more sensitive and specific tests to resolve this controversy.Using this sensitive and specific detection method, we were unable to identify SV40 miRNA expression in human malignant pleural mesothelioma (MM) samples.Our work indicates that SV40 miRNAs are not likely to contribute to mesothelioma tumourogenesis, but highlights the value of this approach when compared with the relatively unspecific current testing methods.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology and Laboratory Medicine, Brown University, Box G-E-5, Providence, RI 02912, USA.

ABSTRACT

Background: Simian virus-40 (SV40) is a DNA tumour virus that was introduced into the human population with contaminated poliovirus vaccine, and its role in mesothelioma is widely debated. PCR based testing has been called into question, as false positives can be because of cross-reactivity with related viruses, or to laboratory contamination. The Institute of Medicine has recommended the development of more sensitive and specific tests to resolve this controversy.

Methods: We have characterized highly sensitive RT-PCR based assays that are specific for SV40-encoded microRNAs (miRNAs), as an alternative to current testing methods.

Results: Using this sensitive and specific detection method, we were unable to identify SV40 miRNA expression in human malignant pleural mesothelioma (MM) samples.

Conclusion: Our work indicates that SV40 miRNAs are not likely to contribute to mesothelioma tumourogenesis, but highlights the value of this approach when compared with the relatively unspecific current testing methods.

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Related in: MedlinePlus

Relative background detection in SV40-transformed cell lines. Assays against the various miRNAs were tested for fidelity using the mouse SV40-transformed cell line, SV-T2, and the human SV40-transformed cell line, SVG-A. Cell lysates enriched for small RNAs were reverse transcribed using each specific assay. Quantitative PCR was performed to measure the levels of each SV40 miRNA in relation to the mouse control small RNA, snoRNA202, and the human control small RNA, RNU48. Error bars represent the s.e. of two experiments for SV-T2 cells or three experiments for SVG-A cells. The relative quantification of miRNA was performed according to the 2−ΔΔCt method, comparing product amplified from mouse cells relative to snoRNA202, and product amplified from human cells relative to RNU48.
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fig2: Relative background detection in SV40-transformed cell lines. Assays against the various miRNAs were tested for fidelity using the mouse SV40-transformed cell line, SV-T2, and the human SV40-transformed cell line, SVG-A. Cell lysates enriched for small RNAs were reverse transcribed using each specific assay. Quantitative PCR was performed to measure the levels of each SV40 miRNA in relation to the mouse control small RNA, snoRNA202, and the human control small RNA, RNU48. Error bars represent the s.e. of two experiments for SV-T2 cells or three experiments for SVG-A cells. The relative quantification of miRNA was performed according to the 2−ΔΔCt method, comparing product amplified from mouse cells relative to snoRNA202, and product amplified from human cells relative to RNU48.

Mentions: We next used SV40-transformed mouse (SV-T2) and human (SVG-A) cell lines to test the sensitivity of all five assays compared with the species-specific small RNAs, snoRNA202 (mouse) and RNU48 (human). These cells contain at least one copy of the entire genome but do not express the late transcript and therefore should not express the mature miRNAs (Botchan et al, 1976; Major and Matsumura, 1984). We compared the background detection of each assay in small RNA-enriched lysates of both cell types and found that both the 5′ and 3′ assays detected <1% of the relative control (Figure 2). The minimal assays had the least cross-reactivity in transformed cell lines with the 3′ and 5′ assays being specific to 100 f and 300 f, respectively.


SV40 associated miRNAs are not detectable in mesotheliomas.

Gee GV, Stanifer ML, Christensen BC, Atwood WJ, Ugolini D, Bonassi S, Resnick MB, Nelson HH, Marsit CJ, Kelsey KT - Br. J. Cancer (2010)

Relative background detection in SV40-transformed cell lines. Assays against the various miRNAs were tested for fidelity using the mouse SV40-transformed cell line, SV-T2, and the human SV40-transformed cell line, SVG-A. Cell lysates enriched for small RNAs were reverse transcribed using each specific assay. Quantitative PCR was performed to measure the levels of each SV40 miRNA in relation to the mouse control small RNA, snoRNA202, and the human control small RNA, RNU48. Error bars represent the s.e. of two experiments for SV-T2 cells or three experiments for SVG-A cells. The relative quantification of miRNA was performed according to the 2−ΔΔCt method, comparing product amplified from mouse cells relative to snoRNA202, and product amplified from human cells relative to RNU48.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2966630&req=5

fig2: Relative background detection in SV40-transformed cell lines. Assays against the various miRNAs were tested for fidelity using the mouse SV40-transformed cell line, SV-T2, and the human SV40-transformed cell line, SVG-A. Cell lysates enriched for small RNAs were reverse transcribed using each specific assay. Quantitative PCR was performed to measure the levels of each SV40 miRNA in relation to the mouse control small RNA, snoRNA202, and the human control small RNA, RNU48. Error bars represent the s.e. of two experiments for SV-T2 cells or three experiments for SVG-A cells. The relative quantification of miRNA was performed according to the 2−ΔΔCt method, comparing product amplified from mouse cells relative to snoRNA202, and product amplified from human cells relative to RNU48.
Mentions: We next used SV40-transformed mouse (SV-T2) and human (SVG-A) cell lines to test the sensitivity of all five assays compared with the species-specific small RNAs, snoRNA202 (mouse) and RNU48 (human). These cells contain at least one copy of the entire genome but do not express the late transcript and therefore should not express the mature miRNAs (Botchan et al, 1976; Major and Matsumura, 1984). We compared the background detection of each assay in small RNA-enriched lysates of both cell types and found that both the 5′ and 3′ assays detected <1% of the relative control (Figure 2). The minimal assays had the least cross-reactivity in transformed cell lines with the 3′ and 5′ assays being specific to 100 f and 300 f, respectively.

Bottom Line: The Institute of Medicine has recommended the development of more sensitive and specific tests to resolve this controversy.Using this sensitive and specific detection method, we were unable to identify SV40 miRNA expression in human malignant pleural mesothelioma (MM) samples.Our work indicates that SV40 miRNAs are not likely to contribute to mesothelioma tumourogenesis, but highlights the value of this approach when compared with the relatively unspecific current testing methods.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology and Laboratory Medicine, Brown University, Box G-E-5, Providence, RI 02912, USA.

ABSTRACT

Background: Simian virus-40 (SV40) is a DNA tumour virus that was introduced into the human population with contaminated poliovirus vaccine, and its role in mesothelioma is widely debated. PCR based testing has been called into question, as false positives can be because of cross-reactivity with related viruses, or to laboratory contamination. The Institute of Medicine has recommended the development of more sensitive and specific tests to resolve this controversy.

Methods: We have characterized highly sensitive RT-PCR based assays that are specific for SV40-encoded microRNAs (miRNAs), as an alternative to current testing methods.

Results: Using this sensitive and specific detection method, we were unable to identify SV40 miRNA expression in human malignant pleural mesothelioma (MM) samples.

Conclusion: Our work indicates that SV40 miRNAs are not likely to contribute to mesothelioma tumourogenesis, but highlights the value of this approach when compared with the relatively unspecific current testing methods.

Show MeSH
Related in: MedlinePlus