Validation of putative reference genes for normalization of Q-RT-PCR data from paraffin-embedded lymphoid tissue

Tina Marie Green, Karin de Stricker, Michael Boe Møller

Publikation: Bidrag til tidsskriftTidsskriftartikelForskningpeer review

Resumé

Udgivelsesdato: 2009-Dec
OriginalsprogEngelsk
TidsskriftDiagnostic Molecular Pathology
Vol/bind18
Udgave nummer4
Sider (fra-til)243-9
Antal sider6
ISSN1052-9551
DOI
StatusUdgivet - 1. dec. 2009

Fingeraftryk

Lymphoid Tissue
Paraffin
Polymerase Chain Reaction
Formaldehyde
Lymph Nodes
TATA-Box Binding Protein
Lymphoma, Large B-Cell, Diffuse
Essential Genes
Lymphoma
Leukemia

Citer dette

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title = "Validation of putative reference genes for normalization of Q-RT-PCR data from paraffin-embedded lymphoid tissue",
abstract = "Normalization of quantitative reverse transcription-PCR (Q-RT-PCR) data to appropriate tissue-specific reference genes is an essential part of interpreting the results. This study aimed to determine the most appropriate reference genes for normalizing gene expressions in lymphatic tissue, represented by non-neoplastic lymph nodes and diffuse large B-cell lymphomas, by using 2 statistical software applications, geNorm and NormFinder. In addition, we wanted to validate the usefulness of paraffin-embedded samples for Q-RT-PCR studies by investigating gene expressions of relevant target genes in paired frozen and paraffin-embedded samples. Moreover, we studied the impact of amplicon sizes on the efficiency of Q-RT-PCR in paraffin-embedded tissues. Six putative reference genes were tested for stability of expression in 21 pairs of snap-frozen and formalin-fixed, paraffin-embedded lymph nodes and lymphomas. The genes were ranked according to their suitability as reference genes. According to both statistical approaches, beta-glucoronidase was the single most appropriate reference gene in both snap-frozen and paraffin-embedded samples. TATA box-binding protein gene and Abelson murine leukemia viral oncogene homolog 1 gene were also highly ranked by both programs. In addition, we measured the relative expressions of 7 target genes by Q-RT-PCR, using PCR primer-probes with amplicon sizes up to 105 bases. The correlation coefficient for expression measured in matched frozen and paraffin-embedded samples was 0.93 (P<0.01) after normalization with the appropriate reference genes. Thus, we show that formalin-fixed, paraffin-embedded lymphoid samples are suitable for Q-RT-PCR when using thoroughly validated reference genes.",
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Validation of putative reference genes for normalization of Q-RT-PCR data from paraffin-embedded lymphoid tissue. / Green, Tina Marie; de Stricker, Karin; Møller, Michael Boe.

I: Diagnostic Molecular Pathology, Bind 18, Nr. 4, 01.12.2009, s. 243-9.

Publikation: Bidrag til tidsskriftTidsskriftartikelForskningpeer review

TY - JOUR

T1 - Validation of putative reference genes for normalization of Q-RT-PCR data from paraffin-embedded lymphoid tissue

AU - Green, Tina Marie

AU - de Stricker, Karin

AU - Møller, Michael Boe

PY - 2009/12/1

Y1 - 2009/12/1

N2 - Normalization of quantitative reverse transcription-PCR (Q-RT-PCR) data to appropriate tissue-specific reference genes is an essential part of interpreting the results. This study aimed to determine the most appropriate reference genes for normalizing gene expressions in lymphatic tissue, represented by non-neoplastic lymph nodes and diffuse large B-cell lymphomas, by using 2 statistical software applications, geNorm and NormFinder. In addition, we wanted to validate the usefulness of paraffin-embedded samples for Q-RT-PCR studies by investigating gene expressions of relevant target genes in paired frozen and paraffin-embedded samples. Moreover, we studied the impact of amplicon sizes on the efficiency of Q-RT-PCR in paraffin-embedded tissues. Six putative reference genes were tested for stability of expression in 21 pairs of snap-frozen and formalin-fixed, paraffin-embedded lymph nodes and lymphomas. The genes were ranked according to their suitability as reference genes. According to both statistical approaches, beta-glucoronidase was the single most appropriate reference gene in both snap-frozen and paraffin-embedded samples. TATA box-binding protein gene and Abelson murine leukemia viral oncogene homolog 1 gene were also highly ranked by both programs. In addition, we measured the relative expressions of 7 target genes by Q-RT-PCR, using PCR primer-probes with amplicon sizes up to 105 bases. The correlation coefficient for expression measured in matched frozen and paraffin-embedded samples was 0.93 (P<0.01) after normalization with the appropriate reference genes. Thus, we show that formalin-fixed, paraffin-embedded lymphoid samples are suitable for Q-RT-PCR when using thoroughly validated reference genes.

AB - Normalization of quantitative reverse transcription-PCR (Q-RT-PCR) data to appropriate tissue-specific reference genes is an essential part of interpreting the results. This study aimed to determine the most appropriate reference genes for normalizing gene expressions in lymphatic tissue, represented by non-neoplastic lymph nodes and diffuse large B-cell lymphomas, by using 2 statistical software applications, geNorm and NormFinder. In addition, we wanted to validate the usefulness of paraffin-embedded samples for Q-RT-PCR studies by investigating gene expressions of relevant target genes in paired frozen and paraffin-embedded samples. Moreover, we studied the impact of amplicon sizes on the efficiency of Q-RT-PCR in paraffin-embedded tissues. Six putative reference genes were tested for stability of expression in 21 pairs of snap-frozen and formalin-fixed, paraffin-embedded lymph nodes and lymphomas. The genes were ranked according to their suitability as reference genes. According to both statistical approaches, beta-glucoronidase was the single most appropriate reference gene in both snap-frozen and paraffin-embedded samples. TATA box-binding protein gene and Abelson murine leukemia viral oncogene homolog 1 gene were also highly ranked by both programs. In addition, we measured the relative expressions of 7 target genes by Q-RT-PCR, using PCR primer-probes with amplicon sizes up to 105 bases. The correlation coefficient for expression measured in matched frozen and paraffin-embedded samples was 0.93 (P<0.01) after normalization with the appropriate reference genes. Thus, we show that formalin-fixed, paraffin-embedded lymphoid samples are suitable for Q-RT-PCR when using thoroughly validated reference genes.

U2 - 10.1097/PDM.0b013e3181a06f42

DO - 10.1097/PDM.0b013e3181a06f42

M3 - Journal article

VL - 18

SP - 243

EP - 249

JO - Diagnostic Molecular Pathology

JF - Diagnostic Molecular Pathology

SN - 1052-9551

IS - 4

ER -