Akademik Veri Yönetim Sistemi
13. ULUSAL MOLEKÜLER BİYOLOJİ VE BİYOTEKNOLOJİ KONGRESİ, 1 - 03 Kasım 2024, ss.20-21, (Özet Bildiri)
Standardization of RNA Concentration in BCR-ABL Analyses: An Evaluation of Accuracy and Repeatability
Abstract
The quantitative determination of BCR-ABL copy numbers is
one of the most critical criteria for
diagnosing and assessing treatment response in hematological
malignancies such as Chronic Myeloid Leukemia (CML). In this test,
standardization contributes to making the tests more consistent, comparable,
and reliable while reducing the occurrence of false positive or negative
results.
This study aims to evaluate the variability of RNA
concentrations between samples for the stan-
dardization of BCR-ABL fusion transcript measurements
concerning the accuracy, sensitivity, and repeatability of the tests.
In our study, the effect of initial RNA quantity on results
was investigated using a simultaneous
quantitative PCR method (Real-Time PCR: RT-PCR) in 20
samples with varying BCR-ABL fusion transcript levels. Single-reaction RT-PCRs
(cDNA synthesis + amplification) were performed using 5 μL of sample and 1000
ng of RNA, regardless of the RNA quantity. A standard curve was generated to
determine BCR-ABL quantities, utilizing 6 different standards for BCR-ABL (106,
105, 104, 103, 102, 101) and 4 different standards for ABL (106, 105, 104, 103)
for absolute quantitative analysis. According to the international scale
standards for BCR-ABL, a minimum of 10,000 ABL copies must be reached;
therefore, samples with fewer than 10,000 ABL copies or those that did not
yield results were excluded from evaluation.
When comparing two different RNA amounts under the same
RT-PCR conditions, 98% of the samples yielded BCR-ABL quantitation when
starting with 1000 ng of RNA. In RT-PCR experiments using 5 μL of RNA, it was
observed that the majority of unassessable samples had low ABL copy values or
did not work at all. When these samples were repeated with an RNA amount of 1000
ng, results were obtained in 80% of cases.
Starting with a standardized RNA amount in BCR-ABL fusion
transcript measurements using the RT-PCR method reduced variability, while high
RNA concentrations negatively affected cDNA synthesis and RT-PCR reactions by
inhibiting them. The standardization of RNA concentration was found to be
crucial for enhancing the accuracy and repeatability of results