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 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.
Dakhama
A
A
University of British Columbia, Pulmonary Research Laboratory, St. Paul's Hospital, Vancouver, Canada.
Macek
V
V
Hogg
J C
JC
Hegele
R G
RG
D000199:Actins / Q000235:genetics*; D016477:Artifacts; D001482:Base Composition; D001483:Base Sequence; D015415:Biomarkers; D015139:Blotting, Southern; D017931:DNA Primers; D004273:DNA, Neoplasm / Q000032:analysis* / Q000235:genetics; D005557:Formaldehyde; D006801:Humans; D008175:Lung Neoplasms / Q000737:chemistry / Q000235:genetics / Q000473:pathology*; D009363:Neoplasm Proteins / Q000235:genetics*; D009691:Nucleic Acid Denaturation; D016612:Paraffin Embedding; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000032:analysis* / Q000737:chemistry; D012334:RNA, Neoplasm / Q000032:analysis* / Q000737:chemistry; D013997:Time Factors; D014020:Tissue Extracts / Q000737:chemistry; D016707:Tissue Fixation
D000199:Actins; D015415:Biomarkers; D017931:DNA Primers; D004273:DNA, Neoplasm; D009363:Neoplasm Proteins; D012333:RNA, Messenger; D012334:RNA, Neoplasm; D014020:Tissue Extracts; D005557:Formaldehyde