FISH Analysis of RB1 Gene in Retinoblastoma

The Cancer Center Core Cytogenetic Laboratory received 10 formalin fixed paraffin embedded (FFPE) retinoblastoma tissue specimens in order to determine by FISH if there is a disruption of the RB1 gene in these tumors. Purified BAC DNA from two RB1 3-prime clones (RPH-115122 and RP11-90K7) were labeled with a green-dUTP (AF488, Molecular Probes) by nick translation, and one RB1 5-prime clone (RP11-795F23) was labeled with a red-dUTP (AF 594, Molecular Probes). In normal cells that contain normal RB1 gene this probe set produces very tightly linked red and green signals since the probes are separated by only 80kb. This assay was specifically designed to detect any disruption of the RB1 gene occurring between introns 6 and 16. One hundred interphase nuclei from each tumor were scored for the presence of either normal RB1 genes (tightly linked red and green signals) or disrupted or deleted RB1 genes (separated red and green signals or deletion of either one). Details for FISH protocol have previously been described [8 (link)].

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McEvoy J., Nagahawatte P., Finkelstein D., Richards-Yutz J., Valentine M., Ma J., Mullighan C., Song G., Chen X., Wilson M., Brennan R., Pounds S., Becksfort J., Huether R., Lu C., Fulton R.S., Fulton L.L., Hong X., Dooling D.J., Ochoa K., Mardis E.R., Wilson R.K., Easton J., Zhang J., Downing J.R., Ganguly A, & Dyer M.A. (2014). RB1 gene inactivation by chromothripsis in human retinoblastoma. Oncotarget, 5(2), 438-450.

Publication 2014

AF 594

A gene Assay Cancer Clone Deletion Dutp Embedded paraffin Fish Formalin Genes Interphase Introns Molecular probes Normal cells Nuclei Rp11 Tumor

Corresponding Organization :

Other organizations : St. Jude Children's Research Hospital, University of Pennsylvania, University of Tennessee Health Science Center, Washington University in St. Louis, Howard Hughes Medical Institute

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Variable analysis

independent variables

Labeling of BAC DNA clones from the RB1 gene with different fluorescent dyes (green-dUTP for RPH-115122 and RP11-90K7, red-dUTP for RP11-795F23)

dependent variables

Presence or absence of tightly linked red and green signals (indicating normal RB1 gene) or separated red and green signals (indicating disruption or deletion of the RB1 gene) in 100 interphase nuclei from each tumor specimen

control variables

Formalin fixed paraffin embedded (FFPE) retinoblastoma tissue specimens
FISH assay specifically designed to detect disruption of the RB1 gene between introns 6 and 16

controls

Positive control: Normal cells containing the RB1 gene, which should show tightly linked red and green signals
Negative control: Not explicitly mentioned

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