Abstract
Molecular signatures of tumors with prognostic and predictive value are now available. However, several technical problems prevent the performing of gene expression profiles in routine diagnostics. The highly sensitive fluorescence-based real-time quantitative reverse transcriptase-polymerase chain reaction procedure is able to analyze mRNA levels from formalin-fixed paraffin-embedded (FFPE) tissues, the most commonly available source of tumor samples with well-documented information. However, the time-dependent fragmentation and small amount of RNA extracted from FFPE requires a preliminary mRNA amplification step to amplify small amounts of mRNA without significantly distorting relative mRNA levels. The purpose of this study was to determine the performance of a commercial cDNA preamplification kit (TaqMan PreAmp Master Mix Kit) on RNA samples obtained from FFPE tissues, prepared, and archived for routine diagnosis. We tested the reliability and reproducibility of this procedure for performing low-density gene expression assay from FFPE tissue samples. The whole procedure reported herein is a powerful and reproducible approach for routine clinical purposes that can be performed even using poorer RNA quality samples and that allows the analysis of gene expression for 96 genes in stored samples.
| Original language | English |
|---|---|
| Pages (from-to) | 112-118 |
| Number of pages | 7 |
| Journal | Diagnostic Molecular Pathology |
| Volume | 18 |
| Issue number | 2 |
| DOIs | |
| Publication status | Published - Jun 2009 |
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Keywords
- Formalin-fixed paraffin-embedded tissue
- Low-density gene expression assay
- Real-time quantitative reverse transcriptase-polymerase chain reaction
- RNA
ASJC Scopus subject areas
- Cell Biology
- Molecular Biology
- Medicine(all)
- Pathology and Forensic Medicine
Cite this
Reliability and reproducibility of a RNA preamplification method for low-density array analysis from formalin-fixed paraffin-embedded breast cancer samples. / Ciotti, Paola; Garuti, Anna; Ballestrero, Alberto; Cirmena, Gabriella; Chiaramondia, Maurizio; Baccini, Paola; Bellone, Emilia; Mandich, Paola.
In: Diagnostic Molecular Pathology, Vol. 18, No. 2, 06.2009, p. 112-118.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Reliability and reproducibility of a RNA preamplification method for low-density array analysis from formalin-fixed paraffin-embedded breast cancer samples
AU - Ciotti, Paola
AU - Garuti, Anna
AU - Ballestrero, Alberto
AU - Cirmena, Gabriella
AU - Chiaramondia, Maurizio
AU - Baccini, Paola
AU - Bellone, Emilia
AU - Mandich, Paola
PY - 2009/6
Y1 - 2009/6
N2 - Molecular signatures of tumors with prognostic and predictive value are now available. However, several technical problems prevent the performing of gene expression profiles in routine diagnostics. The highly sensitive fluorescence-based real-time quantitative reverse transcriptase-polymerase chain reaction procedure is able to analyze mRNA levels from formalin-fixed paraffin-embedded (FFPE) tissues, the most commonly available source of tumor samples with well-documented information. However, the time-dependent fragmentation and small amount of RNA extracted from FFPE requires a preliminary mRNA amplification step to amplify small amounts of mRNA without significantly distorting relative mRNA levels. The purpose of this study was to determine the performance of a commercial cDNA preamplification kit (TaqMan PreAmp Master Mix Kit) on RNA samples obtained from FFPE tissues, prepared, and archived for routine diagnosis. We tested the reliability and reproducibility of this procedure for performing low-density gene expression assay from FFPE tissue samples. The whole procedure reported herein is a powerful and reproducible approach for routine clinical purposes that can be performed even using poorer RNA quality samples and that allows the analysis of gene expression for 96 genes in stored samples.
AB - Molecular signatures of tumors with prognostic and predictive value are now available. However, several technical problems prevent the performing of gene expression profiles in routine diagnostics. The highly sensitive fluorescence-based real-time quantitative reverse transcriptase-polymerase chain reaction procedure is able to analyze mRNA levels from formalin-fixed paraffin-embedded (FFPE) tissues, the most commonly available source of tumor samples with well-documented information. However, the time-dependent fragmentation and small amount of RNA extracted from FFPE requires a preliminary mRNA amplification step to amplify small amounts of mRNA without significantly distorting relative mRNA levels. The purpose of this study was to determine the performance of a commercial cDNA preamplification kit (TaqMan PreAmp Master Mix Kit) on RNA samples obtained from FFPE tissues, prepared, and archived for routine diagnosis. We tested the reliability and reproducibility of this procedure for performing low-density gene expression assay from FFPE tissue samples. The whole procedure reported herein is a powerful and reproducible approach for routine clinical purposes that can be performed even using poorer RNA quality samples and that allows the analysis of gene expression for 96 genes in stored samples.
KW - Formalin-fixed paraffin-embedded tissue
KW - Low-density gene expression assay
KW - Real-time quantitative reverse transcriptase-polymerase chain reaction
KW - RNA
UR - http://www.scopus.com/inward/record.url?scp=67649961336&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=67649961336&partnerID=8YFLogxK
U2 - 10.1097/PDM.0b013e3181831320
DO - 10.1097/PDM.0b013e3181831320
M3 - Article
VL - 18
SP - 112
EP - 118
JO - Diagnostic Molecular Pathology
JF - Diagnostic Molecular Pathology
SN - 1052-9551
IS - 2
ER -