Amplification of human beta-actin gene by the reverse transcriptase-polymerase chain reaction: implications for assessment of RNA from formalin-fixed, paraffin-embedded material

The polymerase chain reaction (PCR) is a powerful method that allows enzymatic amplification of rate target nucleic acid sequences. It has been applied to the amplification of viral genomes from paraffin-embedded pathology specimens. However, interpretation of negative results requires amplification...

Celý popis

Uloženo v:
Podrobná bibliografie
Vydáno v:The journal of histochemistry and cytochemistry Ročník 44; číslo 10; s. 1205
Hlavní autoři: Dakhama, A, Macek, V, Hogg, J C, Hegele, R G
Médium: Journal Article
Jazyk:angličtina
Vydáno: United States 01.10.1996
Témata:
Actins - genetics
Base Composition
Base Sequence
Biomarkers
Blotting, Southern
DNA Primers
DNA, Neoplasm - analysis
DNA, Neoplasm - genetics
Formaldehyde
Humans
Lung Neoplasms - chemistry
Lung Neoplasms - genetics
Lung Neoplasms - pathology
Neoplasm Proteins - genetics
Nucleic Acid Denaturation
Paraffin Embedding
Polymerase Chain Reaction
RNA, Messenger - analysis
RNA, Messenger - chemistry
RNA, Neoplasm - analysis
RNA, Neoplasm - chemistry
Time Factors
Tissue Extracts - chemistry
Tissue Fixation
ISSN:0022-1554
On-line přístup:Zjistit podrobnosti o přístupu
Tagy: Přidat tag
Žádné tagy, Buďte první, kdo vytvoří štítek k tomuto záznamu!
Popis
Shrnutí:The polymerase chain reaction (PCR) is a powerful method that allows enzymatic amplification of rate target nucleic acid sequences. It has been applied to the amplification of viral genomes from paraffin-embedded pathology specimens. However, interpretation of negative results requires amplification of a housekeeping gene such as beta-actin. In the present study we used specific oligonucleotide primers previously designed to amplify both the genomic DNA and the mRNA transcript from paraffin-embedded tissue. These products have predicted sizes of 250 BP and 154 BP, respectively, but our results showed that PCR amplification only (without reverse transcription) unexpectedly generated the 154-BP product. Further investigation of the nature of this product demonstrated that it originated from the amplification of DNA, not RNA. We conclude that the 154-BP product generated by these primers cannot be exclusively considered as beta-actin RNA product and should not be used to assess successful extraction of RNA, to ascertain its integrity, or to normalize for the total amount of RNA assayed by RT-PCR from paraffin-embedded tissue.
Bibliografie:ObjectType-Article-1
SourceType-Scholarly Journals-1
ObjectType-Feature-2
content type line 23
ISSN:0022-1554
DOI:10.1177/44.10.8813086