In situ hybridization: a routine method for parallel localization of DNA sequences and of their transcripts in consecutive paraffin sections with the use of 3H-labelled nick translated cloned DNA probes.
A routine in situ hybridization method is described and discussed, which allows parallel detection of repeated DNA sequences and of their abundant transcripts in consecutive tissue sections of a same biological sample with a unique probe. The protocol, based on the use of classical 3H-labelled nick translated cloned DNA probes and of conventional paraffin sections of ethanol-acetic acid-fixed tissues, consists of a simple combination of procedures in current use for separate detection of either RNA or DNA. Different treatments recommended in other methods are omitted or simplified, making the protocol suitable for routine use. The method is successfully applied here to a test-system where ribosomal sequences are sought in the ovarian follicles of the Lepidopteran Ephestia kühniella, by using a recombinant plasmid containing a Drosophila melanogaster rDNA repeating unit as a probe. Specific and reproducible results are obtained. Sensitivity is sufficient though moderate specific activities are used. Background level is very low. The regionalized distribution of sequences of both types in the chosen model allows to demonstrate that specific detection of RNA requires the systematic removal of DNA from the tissue sections prior to hybridization.
Guelin
M
M
Kejzlarovà-Lepesant
J
J
Lepesant
J A
JA
D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D004275:DNA, Ribosomal / Q000032:analysis*; D003850:Deoxyribonuclease I / Q000378:metabolism; D004331:Drosophila melanogaster; D005260:Female; D007915:Lepidoptera; D008722:Methods; D009693:Nucleic Acid Hybridization; D010053:Ovary / Q000648:ultrastructure; D010957:Plasmids; D014176:Protein Biosynthesis; D012335:RNA, Ribosomal / Q000032:analysis*; D014158:Transcription, Genetic
D004275:DNA, Ribosomal; D012335:RNA, Ribosomal; D003850:Deoxyribonuclease I