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

Dakhama, Azzeddine; Macek, V; Hogg, J C; Hegele, Richard G.

doi:10.1177/44.10.8813086

Cited by 26 publications

(21 citation statements)

References 5 publications

(5 reference statements)

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“…A potential limitation of this approach is the possible degradation of nucleic acids in the heart samples due to fixation and long storage times [19,39,40]. The degradation of RNA is known to occur more easily than that of DNA [40].…”

Section: Discussionmentioning

confidence: 99%

Cytomegalovirus Infection of the Heart Is Common in Patients with Fatal Myocarditis

Kytö

Vuorinen

Saukko

et al. 2005

Clinical Infectious Diseases

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Section: Discussionmentioning

confidence: 99%

Cytomegalovirus Infection of the Heart Is Common in Patients with Fatal Myocarditis

Kytö

Vuorinen

Saukko

et al. 2005

Clinical Infectious Diseases

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“…Paraffin-embedded liver tissue was cut into 10-µm sections and the slides were cleared with xylene. DNA was then extracted using the same kit as for the serum samples and its quality was tested by PCR amplification of the ß actin gene as described by Dakhama et al (30).…”

Section: Hepatitis B Virus Detectionmentioning

confidence: 99%

Low occurrence of occult hepatitis B virus infection and high frequency of hepatitis C virus genotype 3 in hepatocellular carcinoma in Brazil

Alencar

Gomes

Sitnik

et al. 2007

Braz J Med Biol Res

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Occult hepatitis B virus (HBV) infection has been reported among patients with hepatitis C virus (HCV) infection and hepatocellular carcinoma (HCC). Our aim was to evaluate the presence of occult HBV infection in patients with HCV-related liver cirrhosis (LC) with or without HCC in São Paulo, Brazil. Serum and liver tissue samples from 50 hepatitis B surface antigennegative patients with HCV-related LC who underwent liver transplantation at the University of São Paulo School of Medicine Hospital from 1993 to 2004 were divided into groups with LC only (N = 33) and with LC plus HCC (N = 17). HBV DNA was assayed for serum and paraffin-embedded liver tissue (tumoral and non-tumoral) using real time PCR and only 1 case with HCC had HBV DNA-positive serum. All liver samples were negative. HCV genotype 3 was detected in 17/39 (43.7%) cases. In conclusion, using a sensitive real time PCR directed to detect HBV variants circulating in Brazil, occult hepatitis B infection was not found among HCV-positive cirrhotic patients and was rarely found among HCV-positive HCC patients. These results are probably related to the low prevalence of HBV infection in our population. Furthermore, we have also shown that HCV genotype 3 is frequently found in Brazilian cirrhotic patients, particularly when they also have HCC. More studies involving a large number of cases should be carried out to confirm these data and to further characterize Brazilian HCV genotype isolates to elucidate genetic features that might be related to its carcinogenic potential.

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“…This is despite widespread evidence that its levels of transcription can vary widely in response to experimental manipulation in human breast epithelial cells (Spanakis 1993), and blastomeres (Krussel et al 1998), as well as in various porcine tissues (Foss et al 1998) and canine myocardium (Carlyle et al 1996). In addition, the presence of pseudogenes interferes with the interpretation of results (Dirnhofer et al 1995, Raff et al 1997, Mutimer et al 1998) and primers commonly used for detecting -actin mRNA amplify DNA also (Dakhama et al 1996).…”

Section: -Actinmentioning

confidence: 99%

Absolute quantification of mRNA using real-time reverse transcription polymerase chain reaction assays

Bustin¹

2000

Journal of Molecular Endocrinology

3,429

2,448

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The reverse transcription polymerase chain reaction (RT-PCR) is the most sensitive method for the detection of low-abundance mRNA, often obtained from limited tissue samples. However, it is a complex technique, there are substantial problems associated with its true sensitivity, reproducibility and specificity and, as a quantitative method, it suffers from the problems inherent in PCR. The recent introduction of fluorescence-based kinetic RT-PCR procedures significantly simplifies the process of producing reproducible quantification of mRNAs and promises to overcome these limitations. Nevertheless, their successful application depends on a clear understanding of the practical problems, and careful experimental design, application and validation remain essential for accurate quantitative measurements of transcription. This review discusses the technical aspects involved, contrasts conventional and kinetic RT-PCR methods for quantitating gene expression and compares the different kinetic RT-PCR systems. It illustrates the usefulness of these assays by demonstrating the significantly different levels of transcription between individuals of the housekeeping gene family, glyceraldehyde-3-phosphate-dehydrogenase (GAPDH).

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

Cited by 26 publications

References 5 publications

Cytomegalovirus Infection of the Heart Is Common in Patients with Fatal Myocarditis

Cytomegalovirus Infection of the Heart Is Common in Patients with Fatal Myocarditis

Low occurrence of occult hepatitis B virus infection and high frequency of hepatitis C virus genotype 3 in hepatocellular carcinoma in Brazil

Absolute quantification of mRNA using real-time reverse transcription polymerase chain reaction assays

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