Amplification of human beta-actin gene by the reverse transcriptase-polymerase chain reaction: implications for assessment of RNA from formalin-fixed, paraffin-embedded material
| Autor: | R G Hegele, Azzeddine Dakhama, J C Hogg, V Macek |
|---|---|
| Rok vydání: | 1996 |
| Předmět: |
Lung Neoplasms
Time Factors Tissue Fixation Histology Nucleic Acid Denaturation Polymerase Chain Reaction law.invention chemistry.chemical_compound law Formaldehyde Humans RNA Messenger RNA Neoplasm Polymerase chain reaction DNA Primers Base Composition Paraffin Embedding Base Sequence Tissue Extracts Chemistry RNA DNA Neoplasm Molecular biology Actins Reverse transcriptase Neoplasm Proteins Reverse transcription polymerase chain reaction Blotting Southern genomic DNA Nucleic acid Anatomy Artifacts Applications of PCR Biomarkers DNA |
| Zdroj: | Journal of Histochemistry & Cytochemistry. 44:1205-1207 |
| ISSN: | 1551-5044 0022-1554 |
| DOI: | 10.1177/44.10.8813086 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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