Use of spontaneously mutated human DNA as competitive internal standard for nucleic acid quantification by reverse transcription-polymerase chain reaction (RT-PCR).
Quantification of gene expression is of increasing interest in many medical sciences. Methods based on reverse transcription-polymerase chain reactions (RT-PCRs) are timesaving and require only very small amounts of RNA. A limiting factor, however, is the significant fluctuation in the efficacy of reverse transcription as well as in the polymerase chain reactions. Various external and internal standards have been suggested for correcting these fluctuations. We describe a novel way of creating an internal standard for assessing the expression of type VII collagen in human cells. The total RNA of a patient with hereditary epidermolysis bullosa dystrophica associated with a homozygous T to A point mutation in type VII collagen gene was reverse transcribed and a 382bp fragment of type VII collagen cDNA containing the mutation was amplified. The mutated cDNA, unlike normal type VII collagen cDNA could be cleaved by EarI endonuclease into 244bp and 138bp fragments. Semiquantitative PCR was performed with the mutated cDNA as internal standard and the studied cDNA sample in the same tube in the presence of alpha 32P-labeled dCTP. The reaction was followed by EarI digestion, electrophoresis on a polyacrylamide gel and exposure to a X-ray film. In conclusion, we describe a timesaving method for creating internal standards for semiquantitative RT-PCR.
Rudnicka
L
L
Department of Dermatology, Medical School, Warsaw, Poland.
Diaz
A
A
Varga
J
J
Christiano
A
A
Uitto
J
J
Jimenez
S A
SA
D001483:Base Sequence; D003094:Collagen / Q000235:genetics / Q000378:metabolism; D004247:DNA / Q000235:genetics; D005347:Fibroblasts / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation; D016133:Polymerase Chain Reaction / Q000379:methods*; D012333:RNA, Messenger / Q000032:analysis* / Q000378:metabolism; D012015:Reference Standards; D012016:Reference Values; D012595:Scleroderma, Systemic / Q000378:metabolism; D014158:Transcription, Genetic; D016212:Transforming Growth Factor beta / Q000494:pharmacology
D012333:RNA, Messenger; D016212:Transforming Growth Factor beta; D003094:Collagen; D004247:DNA