In situ assessment of mRNA accessibility in heterogeneous tissue samples using elongation factor-1 alpha (EF-1 alpha).
Elongation factor-1 alpha (EF-1 alpha) is an evolutionarily highly conserved universal cofactor of protein synthesis in all living cells. In this study, its use as a positive control in situ hybridization assays for specific detection of mRNA sequences was evaluated. Northern blot analysis of various non-neoplastic and neoplastic cultured cells of different stages of confluence, cell shape, and cell cycle status revealed that EF-1 alpha had a lower and more homogeneous expression than did beta-actin. In situ hybridization assays using digoxigenin-labeled riboprobes for the detection of EF-1 alpha mRNA in routinely formalin-fixed, paraffin-embedded tissue sections showed that EF-1 alpha is a suitable positive control in all types of cells. However, variation of protease pretreatments demonstrated distinct and sometimes mutually exclusive digestion conditions for different cell types within the same tissue sample. Our results indicate that detection of EF-1 alpha mRNA is an appropriate internal standard for in situ hybridization assays and that it is useful to control artifacts such as false negatives caused by inappropriate protease pretreatments. The observed variability of optimal protease pretreatments for different cell types within the same tissue section strengthens the importance of a positive control in in situ hybridization assays.
Gruber
A D
AD
Department of Pathology, New York State College of Veterinary Medicine, Cornell University, Ithaca 14853-6401, USA.
Levine
R A
RA
D016475:3T3 Cells; D000199:Actins / Q000235:genetics; D000818:Animals; D001483:Base Sequence; D002417:Cattle; D002460:Cell Line; D017931:DNA Primers / Q000235:genetics; D010450:Endopeptidases; D006801:Humans; D017403:In Situ Hybridization / Q000379:methods* / Q000592:standards; D051379:Mice; D008969:Molecular Sequence Data; D020648:Peptide Elongation Factor 1; D010445:Peptide Elongation Factors / Q000235:genetics*; D016133:Polymerase Chain Reaction; D015347:RNA Probes; D012333:RNA, Messenger / Q000235:genetics* / Q000378:metabolism* / Q000592:standards; D012015:Reference Standards; D014407:Tumor Cells, Cultured
D000199:Actins; D017931:DNA Primers; D020648:Peptide Elongation Factor 1; D010445:Peptide Elongation Factors; D015347:RNA Probes; D012333:RNA, Messenger; D010450:Endopeptidases