Preamplification techniques for real-time RT-PCR analyses of endomyocardial biopsies

Noutsias, Michel; Rohde, Maria; Block, Andrea; Klippert, Katrin; Lettau, Olga; Blunert, Katja; Hummel, Michael; Kühl, Uwe; Lehmkuhl, H.; Hetzer, Roland; Rauch, Ursula; Poller, Wolfgang; Pauschinger, Matthias; Schultheiß, Heinz Peter; Volk, Hans‐Dieter; Kotsch, Katja

doi:10.1186/1471-2199-9-3

Cited by 62 publications

(56 citation statements)

References 35 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Total RNA isolation was performed by the Urine Exfoliated Cell RNA Purification Kit (Fisher Scientific, Waltham, MA, USA), quantified by Nanodrop, pretreated with Dnase I (Invitrogen, Carlsbad, CA, USA) and reverse transcribed with SuperScript III Reverse Transcriptase (Invitrogen). The uniformity test as well as cDNA preamplification was performed according to Noutsias et al 8 …”

Section: Patients Urine Collection Rna Isolation and Preamplificationmentioning

confidence: 99%

Quadriplex model enhances urine-based detection of prostate cancer

Jamaspishvili

Král

Khomeriki

et al. 2011

Prostate Cancer Prostatic Dis

View full text Add to dashboard Cite

BACKGROUND:The major advantages of urine-based assays are their non-invasive character and ability to monitor prostate cancer (CaP) with heterogeneous foci. While the test for the prostate cancer antigen 3 (PCA3) is commercially available, the aim of our research was to test other putative urine markers in multiplex settings (AMACR (a-methylacyl-CoA racemase), EZH2 (enhancer of zeste homolog 2), GOLM1 (golgi membrane protein 1), MSMB (microseminoprotein, b), SPINK1 (serine peptidase inhibitor) and TRPM8 (transient receptor potential cation channel, subfamily M, member 8)). METHODS:Expression of the candidate biomarkers was studied in sedimented urine using quantitative reverse transcriptase polymerase chain reaction in two sets of patients with and without restriction on serum PSA levels. RESULTS:We confirmed that PCA3 is an independent predictor of cancer in the patients without restriction of serum PSA values (set 1, n ¼ 176, PSA ¼ 0.1-587 ng ml -1 ). However, AMACR was the only parameter that differentiated CaP from nonCaP patients with serum PSA between 3 and 15 ng ml -1 (set 2, n ¼ 104). The area under curve (AUC) for this gene was 0.645 with both sensitivity and specificity at 65%. Further improvement was achieved by multivariate logistic regression analysis, which identified novel duplex (TRPM8 and MSMB), triplex (plus AMACR) and quadriplex (plus PCA3) models for the detection of early CaPs (AUC ¼ 0.665, 0.726 and 0.741, respectively). CONCLUSIONS:Novel quadriplex test could be implemented as an adjunct to serum PSA or urine PCA3 and this could improve decision making for diagnostics in the case of 'PSA dilemma' patients.

show abstract

Section: Patients Urine Collection Rna Isolation and Preamplificationmentioning

confidence: 99%

Quadriplex model enhances urine-based detection of prostate cancer

Jamaspishvili

Král

Khomeriki

et al. 2011

Prostate Cancer Prostatic Dis

View full text Add to dashboard Cite

show abstract

“…Translating this technique to the evaluation of human oocytes is promising; although the human oocyte is smaller (120 mm diameter compared to 180 mm), the polar body is comparable or larger than the starfish polar body (Veeck, 1990). Technological advances including incorporation of non-biased amplification of mRNA may permit clinical analysis of human oocyte gene expression from a single polar body (Noutsias et al, 2008). Such a method may aid with the assessment of oocyte quality and developmental competence in patients using assisted reproductive technologies.…”

mentioning

confidence: 99%

Detection of oocyte mRNA in starfish polar bodies

Klatsky

Carson

Wessel

2010

Molecular Reproduction Devel

View full text Add to dashboard Cite

“…Although normalization of qRT-PCR-derived expression levels requires an appropriate reference or housekeeping gene (HKG) for compensating for differences between samples [15,16], HKG validation is required for each tissue type [17]. Furthermore, low transcript amounts represent an obstacle for simultaneous analysis of multiple targets or lowexpressing genes, and thus, several methods exist for amplifying mRNA transcripts extracted from tissue samples [18][19][20][21]. However, none of these parameters have been described or optimized for gene expression analysis in cervical samples.…”

mentioning

confidence: 99%

Gene expression analysis of interferon κ in laser capture microdissected cervical epithelium

DeCarlo

Escott

Werner

et al. 2008

Analytical Biochemistry

View full text Add to dashboard Cite

Optimal sample handling techniques for tissue preparation and storage, RNA extraction and quantification, and target gene detection are crucial for reliable gene expression analysis. Methods for measuring low-expressing genes, such as interferons, in human cervical samples are not described in the scientific literature. To detect interferon mRNA in human cervical samples we obtained normal and dysplastic frozen and formalin-fixed cervical biopsies from colposcopy. Histopathological diagnosis was performed by one pathologist. Cervical keratinocytes were isolated using laser capture microdissection. Immortalized keratinocytes transduced with or devoid of an HPV oncogene were used for initial method development. RNA from samples was extracted and integrity tested to compare tissue storage and extraction methods. The expression of five housekeeping genes was analyzed in cell lines and patient tissue to permit normalization between samples using quantitative real-time polymerase chain reaction. The usefulness of cDNA amplification was assessed for the detection of low-expressing interferon κ in cervical tissue. Here we report optimal tissue storage conditions, RNA extraction, sample normalization, and transcript amplification, as well as the sensitivity of quantitative real-time polymerase chain reaction and laser capture microdissection, for interferon κ detection in cervical tissue. Without these optimized techniques, interferon κ detection would be unattainable in cervical samples. KeywordsCervical keratinocytes; Frozen and formalin-fixed cervical tissue; Housekeeping genes; Interferons; Interferon κ; Laser capture microdissection; Quantitative real-time polymerase chain reaction Quantitative real-time polymerase chain reaction (qRT-PCR) 1 for the molecular analysis of disease is a powerful and widely used tool. Extraction of high-quality RNA for use in gene expression analysis techniques such as reverse transcription (RT), qRT-PCR, and cDNA microarrays is of great importance. Although obtaining high-quality RNA from cell lines is relatively straightforward, the complexity and heterogeneity of human tissue pose considerable challenges for linking gene expression patterns with disease state. Nonetheless,

show abstract

Preamplification techniques for real-time RT-PCR analyses of endomyocardial biopsies

Cited by 62 publications

References 35 publications

Quadriplex model enhances urine-based detection of prostate cancer

Quadriplex model enhances urine-based detection of prostate cancer

Detection of oocyte mRNA in starfish polar bodies

Gene expression analysis of interferon κ in laser capture microdissected cervical epithelium

Contact Info

Product

Resources

About