Interphotoreceptor Retinoid Binding Protein mRNA Analysis by Polymerase Chain Reaction

Abstract

The authors used the polymerase chain reaction (PCR) to detect the mRNA for interphotoreceptor retinoid-binding protein (IRBP/RBP3), a photoreceptor specific protein, in small samples. They carried out these experiments to assess the feasibility of applying this technique to small tumor samples. Surgically excised tumor samples from four enucleations were analyzed. Messenger RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction was followed by phenol-chloroform purification, reverse-transcription and amplification. The primers used were 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 indicated by amplified 234 bp band. These three samples showed a high IRBP protein level by slot blot and RNA for IRBP detected by northern blot. Hematoxylin-eosin staining of one of these samples revealed a well differentiated tumor with numerous Flexner-Wintersteiner rosettes. In the fourth tumor, a poorly differentiated neoplasm, no IRBP mRNA was detected. The authors' results showed a qualitative variation of IRBP mRNA levels, usually related to the histologic differentiation, with IRBP expressed in well differentiated tumors as well as in the normal human retina in contrast to a poorly differentiated tumor with no detectable IRBP. The feasibility of the reverse transcriptase-PCR (RT-PCR) technique to detect IRBP mRNA in small retinoblastoma tumors, was demonstrated.

Key Words:

• Retinoblastoma
• interphotoreceptor retinoid-binding protein
• mRNA
• reverse transcriptase-PCR
• tumor differentiation