Promoter-specific transcription of the IGF2 gene: a novel rapid, non-radioactive and highly sensitive protocol for mRNA analysis.
The human insulin-like growth factor-II (IGF2) is a regulatory peptide which is critical in normal fetal growth. IGF2 gene transcription is controlled by the usage of four promoters P1-P4 of which promoters P2-P4 are genomically imprinted. Disruption of imprinting and the resulting increase of gene dosage have been shown to be implicated in tumor progression in a variety of human tumors. Due to the need for high amounts of tissue material for conventional methods such as Northern blotting or ribonuclease protection assay (RPA), studies on IGF2 expression have most often been limited to the detection of total IGF2 transcript, though different dysregulatory events can be responsible for the abundance of IGF2 mRNA found in many tumors. We established a highly sensitive competitive RT-PCR assay for the four different transcripts of the IGF2 gene with transcript-specific external RNA competitors in which we take advantage of fluorescence-based quantification on a semiautomated sequencer. The amount of total RNA needed is approximately 100 times lower than the amounts required for Northern blotting or RPA, so that even cytological samples can be analyzed. We applied the assay to a series of eleven hepatoblastomas (HB) in which normal adjacent liver tissue could also be analyzed.
Hartmann
W
W
Department of Neuropathology, University of Bonn Medical Center, Germany.
Waha
A
A
Koch
A
A
Albrecht
S
S
Gray
S G
SG
Ekström
T J
TJ
von Schweinitz
D
D
Pietsch
T
T
D002675:Child, Preschool; D017931:DNA Primers / Q000737:chemistry; D005260:Female; D018197:Hepatoblastoma / Q000235:genetics*; D006801:Humans; D007223:Infant; D007335:Insulin-Like Growth Factor II / Q000235:genetics* / Q000378:metabolism; D008099:Liver / Q000378:metabolism; D008113:Liver Neoplasms / Q000235:genetics* / Q000378:metabolism; D008297:Male; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000032:analysis*; D012334:RNA, Neoplasm / Q000032:analysis; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014158:Transcription, Genetic
D017931:DNA Primers; D012333:RNA, Messenger; D012334:RNA, Neoplasm; D007335:Insulin-Like Growth Factor II