Normalizing complementary DNA by quantitative reverse transcriptase- polymerase chain reaction of β2-microglobulin: Molecular monitoring of minimal residual disease in acute promyelocytic leukemia

Massimo Degan, Francesca Tassan Mazzocco, Raffaele Di Francia, Francesca Maria Rossi, Antonio Pinto, Valter Gattei

Research output: Contribution to journalArticle

Abstract

Reverse transcription (RT)-polymerase chain reaction (PCR) raises unique methodological matters that may hamper the reliability of the procedure, especially when results should direct therapeutic decisions. One of these matters is represented by the RT step. The present study shows that differences in complementary DNA (cDNA) preparations purposely containing increasing amounts of retrotranscribed RNA were not disclosed by nonquantitative RT-PCR by two different housekeeping genes, leading to fictitious results when the expression of a given gene was quantitatively assessed. To overcome this problem, the following are proposed: 1) to evaluate the efficiency of RT step through the quantification, by competitive RT-PCR, of the expression levels of the housekeeping gene β2-microglobulin (β2M); 2) to normalize each cDNA preparation to be comprised within I standard deviation of the mean value of β2M absolute level (3.14 ± 1.14 attomoles/μg RNA) found by analyzing 33 cell lines of hematopoietic origin. To validate this strategy in a clinical setting, serial cDNA samples from patients were checked by conventional and quantitative RT-PCR for β2M. Again, only a quantitative evaluation of β2M levels was allowed to unveil significant differences, otherwise undetected, in the efficiency of RT reactions among these cDNA samples. Normalization of samples to obtain cDNA preparations containing comparable β2M levels, eventually led to an increased sensitivity in the detection of PML-RARα fusion transcripts. This approach seems of great value for the monitoring of minimal residual disease in serial patient samples when a tumor-specific marker is available.

Original languageEnglish
Pages (from-to)98-109
Number of pages12
JournalDiagnostic Molecular Pathology
Volume9
Issue number2
DOIs
Publication statusPublished - Jun 2000

Fingerprint

Acute Promyelocytic Leukemia
Residual Neoplasm
Reverse Transcriptase Polymerase Chain Reaction
Reverse Transcription
Complementary DNA
Polymerase Chain Reaction
Essential Genes
RNA
Tumor Biomarkers
Cell Line
Genes

Keywords

  • Competitive RT-PCR
  • Polymerase chain reaction
  • Promyelocytic leukemia
  • Quantitative RT- PCR
  • Reverse transcription

ASJC Scopus subject areas

  • Pathology and Forensic Medicine

Cite this

@article{d6ecf8120cb947e2879df50ee7891457,
title = "Normalizing complementary DNA by quantitative reverse transcriptase- polymerase chain reaction of β2-microglobulin: Molecular monitoring of minimal residual disease in acute promyelocytic leukemia",
abstract = "Reverse transcription (RT)-polymerase chain reaction (PCR) raises unique methodological matters that may hamper the reliability of the procedure, especially when results should direct therapeutic decisions. One of these matters is represented by the RT step. The present study shows that differences in complementary DNA (cDNA) preparations purposely containing increasing amounts of retrotranscribed RNA were not disclosed by nonquantitative RT-PCR by two different housekeeping genes, leading to fictitious results when the expression of a given gene was quantitatively assessed. To overcome this problem, the following are proposed: 1) to evaluate the efficiency of RT step through the quantification, by competitive RT-PCR, of the expression levels of the housekeeping gene β2-microglobulin (β2M); 2) to normalize each cDNA preparation to be comprised within I standard deviation of the mean value of β2M absolute level (3.14 ± 1.14 attomoles/μg RNA) found by analyzing 33 cell lines of hematopoietic origin. To validate this strategy in a clinical setting, serial cDNA samples from patients were checked by conventional and quantitative RT-PCR for β2M. Again, only a quantitative evaluation of β2M levels was allowed to unveil significant differences, otherwise undetected, in the efficiency of RT reactions among these cDNA samples. Normalization of samples to obtain cDNA preparations containing comparable β2M levels, eventually led to an increased sensitivity in the detection of PML-RARα fusion transcripts. This approach seems of great value for the monitoring of minimal residual disease in serial patient samples when a tumor-specific marker is available.",
keywords = "Competitive RT-PCR, Polymerase chain reaction, Promyelocytic leukemia, Quantitative RT- PCR, Reverse transcription",
author = "Massimo Degan and Mazzocco, {Francesca Tassan} and {Di Francia}, Raffaele and Rossi, {Francesca Maria} and Antonio Pinto and Valter Gattei",
year = "2000",
month = "6",
doi = "10.1097/00019606-200006000-00006",
language = "English",
volume = "9",
pages = "98--109",
journal = "Diagnostic Molecular Pathology",
issn = "1052-9551",
publisher = "Lippincott Williams and Wilkins",
number = "2",

}

TY - JOUR

T1 - Normalizing complementary DNA by quantitative reverse transcriptase- polymerase chain reaction of β2-microglobulin

T2 - Molecular monitoring of minimal residual disease in acute promyelocytic leukemia

AU - Degan, Massimo

AU - Mazzocco, Francesca Tassan

AU - Di Francia, Raffaele

AU - Rossi, Francesca Maria

AU - Pinto, Antonio

AU - Gattei, Valter

PY - 2000/6

Y1 - 2000/6

N2 - Reverse transcription (RT)-polymerase chain reaction (PCR) raises unique methodological matters that may hamper the reliability of the procedure, especially when results should direct therapeutic decisions. One of these matters is represented by the RT step. The present study shows that differences in complementary DNA (cDNA) preparations purposely containing increasing amounts of retrotranscribed RNA were not disclosed by nonquantitative RT-PCR by two different housekeeping genes, leading to fictitious results when the expression of a given gene was quantitatively assessed. To overcome this problem, the following are proposed: 1) to evaluate the efficiency of RT step through the quantification, by competitive RT-PCR, of the expression levels of the housekeeping gene β2-microglobulin (β2M); 2) to normalize each cDNA preparation to be comprised within I standard deviation of the mean value of β2M absolute level (3.14 ± 1.14 attomoles/μg RNA) found by analyzing 33 cell lines of hematopoietic origin. To validate this strategy in a clinical setting, serial cDNA samples from patients were checked by conventional and quantitative RT-PCR for β2M. Again, only a quantitative evaluation of β2M levels was allowed to unveil significant differences, otherwise undetected, in the efficiency of RT reactions among these cDNA samples. Normalization of samples to obtain cDNA preparations containing comparable β2M levels, eventually led to an increased sensitivity in the detection of PML-RARα fusion transcripts. This approach seems of great value for the monitoring of minimal residual disease in serial patient samples when a tumor-specific marker is available.

AB - Reverse transcription (RT)-polymerase chain reaction (PCR) raises unique methodological matters that may hamper the reliability of the procedure, especially when results should direct therapeutic decisions. One of these matters is represented by the RT step. The present study shows that differences in complementary DNA (cDNA) preparations purposely containing increasing amounts of retrotranscribed RNA were not disclosed by nonquantitative RT-PCR by two different housekeeping genes, leading to fictitious results when the expression of a given gene was quantitatively assessed. To overcome this problem, the following are proposed: 1) to evaluate the efficiency of RT step through the quantification, by competitive RT-PCR, of the expression levels of the housekeeping gene β2-microglobulin (β2M); 2) to normalize each cDNA preparation to be comprised within I standard deviation of the mean value of β2M absolute level (3.14 ± 1.14 attomoles/μg RNA) found by analyzing 33 cell lines of hematopoietic origin. To validate this strategy in a clinical setting, serial cDNA samples from patients were checked by conventional and quantitative RT-PCR for β2M. Again, only a quantitative evaluation of β2M levels was allowed to unveil significant differences, otherwise undetected, in the efficiency of RT reactions among these cDNA samples. Normalization of samples to obtain cDNA preparations containing comparable β2M levels, eventually led to an increased sensitivity in the detection of PML-RARα fusion transcripts. This approach seems of great value for the monitoring of minimal residual disease in serial patient samples when a tumor-specific marker is available.

KW - Competitive RT-PCR

KW - Polymerase chain reaction

KW - Promyelocytic leukemia

KW - Quantitative RT- PCR

KW - Reverse transcription

UR - http://www.scopus.com/inward/record.url?scp=0034129505&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0034129505&partnerID=8YFLogxK

U2 - 10.1097/00019606-200006000-00006

DO - 10.1097/00019606-200006000-00006

M3 - Article

C2 - 10850546

AN - SCOPUS:0034129505

VL - 9

SP - 98

EP - 109

JO - Diagnostic Molecular Pathology

JF - Diagnostic Molecular Pathology

SN - 1052-9551

IS - 2

ER -