mRNA differential display of human laryngeal carcinoma cells and corresponding benign keratinocytes: cloning versus PCR-amplification.
The mRNA expression profile of mucosal keratinocyte cell lines was compared to that of squamous cell carcinoma cell lines of the head and neck (UMSCC-10A and UTSCC-19A) by the differential display technique. Before the sequencing procedure, the differentially expressed fragments can be reamplified either by PCR amplification only or by PCR amplification combined with molecular cloning. In this study, these two methods are compared. Total RNA of both cell types was reverse transcribed into cDNA followed by amplification with PCR. After electrophoresis in a non-denaturing gel, differentially expressed fragments were isolated, reamplified, and sequenced. Reamplification was carried out following two different protocols: the first one included two additional rounds of PCR, the second one only one additional round of PCR followed by cloning. Differentially expressed fragments could be sequenced after reamplification with both methods. Different cloned recombinants of the same PCR pool showed sequence differences in one to three bases. On the other hand, amplification of the differentially expressed fragment by PCR alone was reproducible without any differences in sequence. But in the latter case, only one of the complementary strands could be sequenced. Differentially expressed mRNA fragments detected by differential display can be sequenced directly after reamplification in two additional rounds of PCR. This method is no adequate replacement for bacterial cloning, but it might be a suitable solution if cloning procedures cannot be performed.
Rathcke
I O
IO
Department of Otorhinolaryngology, Philipps-University Marburg, Germany.
Weber
E H
EH
Goeroegh
T
T
Lippert
B M
BM
Folz
B J
BJ
Werner
J A
JA
D000328:Adult; D002294:Carcinoma, Squamous Cell / Q000235:genetics* / Q000473:pathology*; D003001:Cloning, Molecular / Q000379:methods*; D018076:DNA, Complementary / Q000235:genetics; D006801:Humans; D015603:Keratinocytes / Q000473:pathology*; D007820:Laryngeal Mucosa / Q000473:pathology; D007822:Laryngeal Neoplasms / Q000235:genetics* / Q000473:pathology*; D008297:Male; D008875:Middle Aged; D012333:RNA, Messenger / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D014407:Tumor Cells, Cultured
D018076:DNA, Complementary; D012333:RNA, Messenger