Author/Editor		Dakhama, Azzeddine; Maček, Vasilija; Hogg, James C; Hegele, Richard G
Title		Amplification of human beta-actin gene by the reverse transcriptase-polymerase chain reaction: implications for assessment of RNA from formalin-fixed, paraffin-embedded material
Type		članek
Source		J Histochem Cytochem
Vol. and No.		Letnik 44, št. 10
Publication year		1996
Volume		str. 1205-7
Language		eng
Abstract		The polymerase chain reaction (PCR) is a powerful method that allows enzymatic amplification of rare 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 (i-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.
Descriptors		TISSUE FIXATION ACTINS LUNG NEOPLASMS NEOPLASM PROTEINS PARAFFIN EMBEDDING TISSUE EXTRACTS BASE COMPOSITION TIME FACTORS RNA, NEOPLASM RNA, MESSENGER POLYMERASE CHAIN REACTION BASE SEQUENCE BIOLOGICAL MARKERS BLOTTING, SOUTHERN DNA PRIMERS DNA, NEOPLASM FORMALDEHYDE ARTIFACTS