Linked Life Data

8744725

Source:http://linkedlifedata.com/resource/pubmed/id/8744725

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
2
pubmed:dateCreated
1996-10-16
pubmed:abstractText
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.
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:issn
0004-069X
pubmed:author
pubmed:issnType
Print
pubmed:volume
43
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
111-5
pubmed:dateRevised
2004-11-17
pubmed:meshHeading
pubmed:year
1995
pubmed:articleTitle
Use of spontaneously mutated human DNA as competitive internal standard for nucleic acid quantification by reverse transcription-polymerase chain reaction (RT-PCR).
pubmed:affiliation
Department of Dermatology, Medical School, Warsaw, Poland.
pubmed:publicationType
Journal Article