Molecular Genetic PML - RARA Transcrpit Quantitative Analysis - RT-PCR
Test Code
MOG49
Also Known As
No data available
Test Parameters Included
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Department
MOLECULAR GENETICS
Methodology
RT-PCR (Reverse Transcription Polymerase Chain Reaction), Quantitative / Real-Time RT-PCR (RT-qPCR)
This test utilizes Quantitative Reverse Transcription Polymerase Chain Reaction (RT-qPCR), often referred to as Real-Time RT-PCR. This molecular technique is highly sensitive and specific for detecting and quantifying the PML-RARA fusion gene transcript (mRNA).
Principle of RT-qPCR for PML-RARA:
* RNA Extraction: Total RNA, including mRNA, is first isolated from the patient's blood or bone marrow sample.
* Reverse Transcription (RT): The extracted RNA is then reverse transcribed into complementary DNA (cDNA) using an enzyme called reverse transcriptase. This step is crucial because PCR amplifies DNA, not RNA.
* Real-Time Polymerase Chain Reaction (qPCR): The cDNA is then used as a template for PCR amplification. Specific primers are designed to bind only to the PML-RARA fusion transcript. As DNA amplification occurs, a fluorescent signal is generated and detected in real-time.
* Quantitative Aspect: The "quantitative" aspect means that the amount of fluorescence generated is directly proportional to the initial amount of PML-RARA cDNA (and thus mRNA) in the sample. By comparing the amplification curve to a standard curve, the initial copy number of PML-RARA transcripts can be accurately quantified.
* Breakpoint Detection: The assay can typically detect the three common breakpoints within the PML gene (bcr1, bcr2, and bcr3) that fuse with the RARA gene, providing isoform-specific quantification.
* Internal Control: An internal control gene (most commonly ABL1) is simultaneously amplified and quantified to ensure the quality of the RNA extraction and reverse transcription steps, and to normalize the results, which accounts for variations in sample quality or RNA input. The final result is often reported as a normalized ratio of %PML-RARA copies/ABL1 copies.
RT-qPCR is the gold standard for monitoring minimal residual disease (MRD) in acute promyelocytic leukemia (APL) due to its high sensitivity, allowing detection of very low levels of leukemic cells that might be missed by other methods like conventional cytogenetics or FISH.
Sample Required
Whole Blood (WB) / Bone Marrow Aspirate (BMA) collected in EDTA Vacutainer
* Approximately 3-5 mL of Whole Blood or 1-3 mL of Bone Marrow Aspirate is typically required.
* EDTA (lavender-top) tubes are the preferred anticoagulant as they preserve nucleic acids.
* Samples must be collected and transported properly (e.g., refrigerated, not frozen) and typically arrive at the lab within 48-72 hours of collection due to RNA lability.
Preparation
Proper collection and handling of the sample are paramount for the accuracy and reliability of this molecular test, as RNA is highly susceptible to degradation. * No specific patient preparation is re... Read more
Schedule Report
5 Days
Emergency Report
Yes
Frequently Asked Questions
No data available
Test Description
- High-end laboratory & medical equipment.
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