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 language | English |
|---|---|
| Pages (from-to) | 98-109 |
| Number of pages | 12 |
| Journal | Diagnostic Molecular Pathology |
| Volume | 9 |
| Issue number | 2 |
| DOIs | |
| Publication status | Published - Jun 2000 |
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Keywords
- Competitive RT-PCR
- Polymerase chain reaction
- Promyelocytic leukemia
- Quantitative RT- PCR
- Reverse transcription
ASJC Scopus subject areas
- Pathology and Forensic Medicine
Cite this
Normalizing complementary DNA by quantitative reverse transcriptase- polymerase chain reaction of β2-microglobulin : Molecular monitoring of minimal residual disease in acute promyelocytic leukemia. / Degan, Massimo; Mazzocco, Francesca Tassan; Di Francia, Raffaele; Rossi, Francesca Maria; Pinto, Antonio; Gattei, Valter.
In: Diagnostic Molecular Pathology, Vol. 9, No. 2, 06.2000, p. 98-109.Research output: Contribution to journal › Article
}
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 -