Design and Testing of β-Actin Primers for RT-PCR that Do Not Co-amplify Processed Pseudogenes
Quantitative reverse transcription polymerase chain reaction (RT-PCR) is being used increasingly as an alternative to Northern blots analysis or RNase protection assays for quantitation of gene expression. To quantify different samples, measurements are often normalized using the expression of so-ca...
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| Main Authors: | , , , , |
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| Format: | Article |
| Language: | English |
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Taylor & Francis Group
1997-09-01
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| Series: | BioTechniques |
| Online Access: | https://www.future-science.com/doi/10.2144/97233st02 |
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| _version_ | 1850152519087423488 |
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| author | T. Raff M. van der Giet D. Endemann T. Wiederholt M. Paul |
| author_facet | T. Raff M. van der Giet D. Endemann T. Wiederholt M. Paul |
| author_sort | T. Raff |
| collection | DOAJ |
| description | Quantitative reverse transcription polymerase chain reaction (RT-PCR) is being used increasingly as an alternative to Northern blots analysis or RNase protection assays for quantitation of gene expression. To quantify different samples, measurements are often normalized using the expression of so-called “housekeeping” genes, such as cytoplasmic β-actin or glyceraldehyde-3-phosphate dehydrogenase. This approach can produce false results because the presence of processed pseudogenes in the genome, which are related to some of the commonly used transcripts of housekeeping genes, leads to coamplification of contaminating genomic DNA. By yielding amplification products of the same or similar size as the reverse-transcribed target, mRNA quantitation of expression is prone to error. In this paper, we report the results of using three sets of β-actin primers for RT-PCR in the presence and absence of genomic DNA. In addition, we propose two new pairs of oligonucleotide primers that specifically amplify the human and rat β-actin reverse-transcribed mRNA but not pseudogene sequences. These primers are especially suitable for quantitation of mRNA in small tissue samples (e.g., biopsies), where DNase digestion is not feasible, and therefore DNA contamination cannot be avoided. |
| format | Article |
| id | doaj-art-111cc8ba40b940e28fb50eee45faa1ff |
| institution | OA Journals |
| issn | 0736-6205 1940-9818 |
| language | English |
| publishDate | 1997-09-01 |
| publisher | Taylor & Francis Group |
| record_format | Article |
| series | BioTechniques |
| spelling | doaj-art-111cc8ba40b940e28fb50eee45faa1ff2025-08-20T02:25:58ZengTaylor & Francis GroupBioTechniques0736-62051940-98181997-09-0123345646010.2144/97233st02Design and Testing of β-Actin Primers for RT-PCR that Do Not Co-amplify Processed PseudogenesT. Raff0M. van der Giet1D. Endemann2T. Wiederholt3M. Paul41Universitätsklinikum, Benjamin Franklin, Free University, Berlin1Universitätsklinikum, Benjamin Franklin, Free University, Berlin1Universitätsklinikum, Benjamin Franklin, Free University, Berlin1Universitätsklinikum, Benjamin Franklin, Free University, Berlin1Universitätsklinikum, Benjamin Franklin, Free University, BerlinQuantitative reverse transcription polymerase chain reaction (RT-PCR) is being used increasingly as an alternative to Northern blots analysis or RNase protection assays for quantitation of gene expression. To quantify different samples, measurements are often normalized using the expression of so-called “housekeeping” genes, such as cytoplasmic β-actin or glyceraldehyde-3-phosphate dehydrogenase. This approach can produce false results because the presence of processed pseudogenes in the genome, which are related to some of the commonly used transcripts of housekeeping genes, leads to coamplification of contaminating genomic DNA. By yielding amplification products of the same or similar size as the reverse-transcribed target, mRNA quantitation of expression is prone to error. In this paper, we report the results of using three sets of β-actin primers for RT-PCR in the presence and absence of genomic DNA. In addition, we propose two new pairs of oligonucleotide primers that specifically amplify the human and rat β-actin reverse-transcribed mRNA but not pseudogene sequences. These primers are especially suitable for quantitation of mRNA in small tissue samples (e.g., biopsies), where DNase digestion is not feasible, and therefore DNA contamination cannot be avoided.https://www.future-science.com/doi/10.2144/97233st02 |
| spellingShingle | T. Raff M. van der Giet D. Endemann T. Wiederholt M. Paul Design and Testing of β-Actin Primers for RT-PCR that Do Not Co-amplify Processed Pseudogenes BioTechniques |
| title | Design and Testing of β-Actin Primers for RT-PCR that Do Not Co-amplify Processed Pseudogenes |
| title_full | Design and Testing of β-Actin Primers for RT-PCR that Do Not Co-amplify Processed Pseudogenes |
| title_fullStr | Design and Testing of β-Actin Primers for RT-PCR that Do Not Co-amplify Processed Pseudogenes |
| title_full_unstemmed | Design and Testing of β-Actin Primers for RT-PCR that Do Not Co-amplify Processed Pseudogenes |
| title_short | Design and Testing of β-Actin Primers for RT-PCR that Do Not Co-amplify Processed Pseudogenes |
| title_sort | design and testing of β actin primers for rt pcr that do not co amplify processed pseudogenes |
| url | https://www.future-science.com/doi/10.2144/97233st02 |
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