RETINOBLASTOMA - INTERPHOTORECEPTOR RETINOID-BINDING PROTEIN MESSENGER-RNA ANALYSIS BY POLYMERASE CHAIN-REACTION

The authors used the polymerase chain reaction (PCR) to detect the mRN A for interphotoreceptor retinoid-binding protein (IRBP/RBP3), a photo receptor specific protein, in small samples. They carried out these ex periments to assess the feasibility of applying this technique to smal l tumor samples. Surgically excised tumor samples from four enucleatio ns were analyzed. Messenger RNA isolation by acid guanidinium thiocyan ate-phenol-chloroform extraction was followed by phenol-chloroform pur ification, reverse-transcription and amplification. The primers used w ere 5' TGATGACTCTGTCAGTG 3' in exon 3 (sense) and 5' TTGTGCTGGAGCATCTC 3' in exon 4 (antisense). Controls included an IRBP cDNA pIRBP 20-700 and RNA from normal human retina. All samples amplified the same size band if detected. Three tumor samples contained IRBP mRNA as indicate d by amplified 234 bp band. These three samples showed a high IRBP pro tein level by slot blot and RNA for IRBP detected by northern blot. He matoxylin-eosin staining of one of these samples revealed a well diffe rentiated tumor with numerous Flexner-Wintersteiner rosettes. In the f ourth tumor, a poorly differentiated neoplasm, no IRBP mRNA was detect ed. The authors' results showed a qualitative variation of IRBP mRNA l evels, usually related to the histologic differentiation, with IRBP ex pressed in well differentiated tumors as well as in the normal human r etina in contrast to a poorly differentiated tumor with no detectable IRBP. The feasibility of the reverse transcriptase-PCR (RT-PCR) techni que to detect IRBP mRNA in small retinoblastoma tumors, was demonstrat ed.