Detection of Viral Infection and Gene Expression in Clinical Tissue Specimens Using Branched DNA (bDNA) In Situ Hybridization

Kenny, Daryn A.; Shen, Lu-Ping; Kolberg, Janice A.

doi:10.1177/002215540205000909

Cited by 24 publications

(13 citation statements)

References 25 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The ViewRNA assay is based on bDNA amplification technology, which realizes remarkable signal amplification, while preserving high specificity. Although this product was developed solely for RNA detection, it can also be used to detect DNA (25). All probe sets were custom synthesized by Affymetrix using sequences from an isolate (GenBank: AB675675, genotype C, most prevalent in eastern China), except for HCV (VF1-10121, target sequence JFH-1 NS3-NS5B region).…”

Section: Methodsmentioning

confidence: 99%

“…In this study, we exploited and modified the ViewRNA technology, making it capable of visualizing RNA and DNA. The ViewRNA technology uses the branched DNA (bDNA) amplification scheme, which was reported to achieve single-copy gene detection under optimal conditions, while preserving high specificity (24,25). Most important, this methodology does not rely on enzyme-based nucleic acid amplification, which obviates complications such as cross contamination or diffusion of amplified products.…”

Section: The Merits Of Our Ish Techniquementioning

confidence: 99%

See 1 more Smart Citation

In situ analysis of intrahepatic virological events in chronic hepatitis B virus infection

Zhang¹,

Lu²,

Zheng³

et al. 2016

Journal of Clinical Investigation

107

View full text Add to dashboard Cite

Section: Methodsmentioning

confidence: 99%

Section: The Merits Of Our Ish Techniquementioning

confidence: 99%

In situ analysis of intrahepatic virological events in chronic hepatitis B virus infection

Zhang¹,

Lu²,

Zheng³

et al. 2016

Journal of Clinical Investigation

107

View full text Add to dashboard Cite

“…25,26 In our study, we determined the impact of different protocols for the identification of HPV by FISH with respect to signal distribution throughout the epithelium, signal pattern (diffuse/punctate/…”

Section: -4mentioning

confidence: 99%

“…12 Moreover, HPV RNA can contribute to the signals observed in these (F)ISH protocols. 25,26 In our study, we determined the impact of different protocols for the identification of HPV by FISH with respect to signal distribution throughout the epithelium, signal pattern (diffuse/punctate/ granular) and contribution of HPV RNA to the final hybridization signal. For this purpose we compared a frequently used HPV detection method that employs a mild pretreatment protocol to a protocol that we developed for the visualization of chromosomal targets.…”

mentioning

confidence: 99%

HPV in situ hybridization: Impact of different protocols on the detection of integrated HPV

Hopman

Kamps

Smedts³

et al. 2005

Intl Journal of Cancer

View full text Add to dashboard Cite

Although there is consensus that HPV integration is common in invasive cervical carcinomas and uncommon or absent in lowgrade uterine cervical intraepithelial neoplasia (CIN I), estimates for HPV integration in CIN II/III range from 5 to 100% using different PCR-based and in situ hybridization (ISH) approaches. It has been suggested that HPV integration can be identified using ISH by scoring of punctate signals. The increased sensitivity of fluorescence ISH (FISH) methods, allowing the detection of single copies of HPV, complicates the distinction between integrated and episomal HPV. Recently it has been suggested that, in such assays, the signals originating from integrated virus can be hidden in a background of episomal HPV. We therefore compared 2 different FISH protocols for the detection of integrated HPV in a series of CIN II/III lesions: 1) a mild protocol in which episomal HPV and RNA is retained and 2) a harsh protocol that extensively extracts proteins and RNA, and which promotes the partial loss of episomal HPV but not integrated HPV. A series of 28 HPV 16/18 positive CIN II/III lesions (17 solitary lesions and 11 lesions adjacent to microinvasive carcinoma) were studied. A punctate signal pattern was identified in 7 of these lesions with both protocols. Punctate signal was also present in control samples from lesions that are known to be associated with HPV integration (invasive squamous cell carcinoma (n ¼ 3), adenocarcinoma in situ (n ¼ 3), and invasive adenocarcinoma (n ¼ 1). HPV RNA contributed significantly to the intensity of punctate FISH signal, especially when applying the mild protocol, as shown by omitting DNA denaturation, including RNase pretreatment steps and measuring the fluorescence signal intensity. Also, HPV RNA was frequently detected in addition to episomal/integrated HPV DNA in the majority of the other 21 CIN II/III lesions; this resulted in intense granular/ diffuse FISH signals throughout the epithelium. However, in 7 of these lesions, the harsh protocol gave a more consistent punctate pattern in cells throughout the full thickness of the epithelium. This supports the hypothesis that the harsh protocol unmasks integrated HPV more efficiently by extracting RNA and episomal HPV. Overall, with this harsh protocol, a clonally expanded population of cells containing punctate HPV signals was found in 5 of 17 (29%) solitary CIN II/III lesions and in 9 of 11 (88%) CIN II/ III lesions associated with microinvasive carcinoma. Combining these data with the results from our previous study, with the harsh protocol in 7 of 40 (18%) Several premalignant stages can be distinguished in the development of carcinoma of the uterine cervix. These include cervical intraepithelial neoplasia grades I, II and III (CIN I-III), also designated low-grade squamous intraepithelial lesions (LSIL, comprising CIN I) and high grade SIL (HSIL, comprising CIN II-III). 1-4Nearly all invasive cervical carcinomas (ICCs) and CIN grade II/ III lesions contain human papillomavirus (HPV) DNA. Epidemiological studies ...

show abstract

“…Since DNA FISH does not require strand-specificity, oligonucleotide probes can be designed to target either of the two complementary strands in the target region. Branched DNA probes, where signal amplification is achieved by two sequential rounds of specific and amplifier probes can also be used to enhance the signal-to-noise ratio of FISH signals 20 . Alternatively, RNA probes such as riboprobes can be used although in our DNAbased probes guarantee a better tradeoff between signal quality, specificity and ease of use.…”

Section: Discussionmentioning

confidence: 99%

Transcriptional Analysis by Nascent RNA FISH of <em>In Vivo</em> Trophoblast Giant Cells or <em>In Vitro</em> Short-term Cultures of Ectoplacental Cone Explants

Corbel

Heard

2016

JoVE

View full text Add to dashboard Cite

The placenta derives from one extra-embryonic lineage, the trophectoderm. In the peri-implantation murine blastocyst, mural trophectoderm cells differentiate into primary trophoblast giant cells (TGCs) while the polar trophectoderm overlying the inner cell mass continues to proliferate later differentiating into secondary TGCs. TGCs play a key role in developing placenta and are essential for a successful pregnancy. Investigation of transcriptional regulation of specific genes during post-implantation development can give insights into TGCs development. Cells of the ectoplacental cone (EPC) from embryos at 7-7.5 days of gestation (E7-7.5), derived from the polar trophectoderm, differentiate into secondary TGCs 1 . TGCs can be studied in situ, on cryostat sections of embryos at E7 although the number of TGCs is very low at this stage. An alternative means of analyzing secondary TGCs is to use short-term cultures of individual EPCs from E7 embryos. We propose a technique to investigate the transcriptional status of genes of interest both in vivo and in vitro at the single-cell level using fluorescent in situ hybridization (RNA FISH) to visualize nascent transcripts. This technique provides a direct readout of gene expression and enables assessment of the chromosomal status of TGCs, which are large endoreplicating cells. Indeed, a key feature of terminal differentiation of TGCs is that they exit the cell cycle and undergo multiple rounds of endoreplication.This approach can be applied to detect expression of any gene expressed from autosomes and/or sex chromosomes and can provide important information into developmental mechanisms as well as placental diseases.

show abstract

Detection of Viral Infection and Gene Expression in Clinical Tissue Specimens Using Branched DNA (bDNA) In Situ Hybridization

Cited by 24 publications

References 25 publications

In situ analysis of intrahepatic virological events in chronic hepatitis B virus infection

In situ analysis of intrahepatic virological events in chronic hepatitis B virus infection

HPV in situ hybridization: Impact of different protocols on the detection of integrated HPV

Transcriptional Analysis by Nascent RNA FISH of <em>In Vivo</em> Trophoblast Giant Cells or <em>In Vitro</em> Short-term Cultures of Ectoplacental Cone Explants

Contact Info

Product

Resources

About