Detecting the EWS/FLI1 Fusion Transcript in Ewing’s Cancer: A Guide to using Reverse-Transcriptase PCR (RT-PCR) with Targeted Primers

EWS/ EWINGScancer/solid tumort(11:22)Reverse-Transcriptase PCR (RT-PCR)PCR primers designed to yield product only if translocation has occurred

In the case of Ewing’s cancer, also known as Ewing’s sarcoma, a specific chromosomal translocation known as the EWS/FLI1 fusion is commonly found

In the case of Ewing’s cancer, also known as Ewing’s sarcoma, a specific chromosomal translocation known as the EWS/FLI1 fusion is commonly found. This translocation involves the fusion of the EWS gene on chromosome 22 with the FLI1 gene on chromosome 11, resulting in an abnormal hybrid gene.

Reverse-Transcriptase PCR (RT-PCR) is a molecular technique frequently used to detect gene expression and genetic abnormalities, including gene fusions. In the context of Ewing’s cancer, RT-PCR can be employed to identify the presence of the EWS/FLI1 fusion transcript.

To perform RT-PCR for EWS/FLI1 fusion detection, PCR primers are specifically designed to target the unique sequence created by the fusion of the EWS and FLI1 genes. These primers are designed to yield a PCR product only if the translocation has occurred and the fusion transcript is present.

The process involves the following steps:

1. RNA isolation: Total RNA is extracted from the tumor sample using a suitable method to preserve the RNA integrity.

2. cDNA synthesis: Reverse transcription is performed using a reverse transcriptase enzyme and specific primers to convert the RNA into complementary DNA (cDNA). This cDNA contains the EWS/FLI1 fusion transcript if the translocation has occurred.

3. PCR setup: PCR primers are designed to anneal to regions flanking the junction site of the EWS/FLI1 fusion transcript. These primers should be specific to the fusion sequence and not hybridize with the wild type EWS or FLI1 genes. One primer is designed to anneal to the EWS gene sequence, while the other primer is designed to anneal to the FLI1 gene sequence. These primers are chosen to span the fusion junction site.

4. PCR amplification: The cDNA template from step 2 is mixed with PCR reagents, including DNA polymerase, nucleotides, and a suitable buffer. Cycling conditions are optimized to allow for amplification of the specific target sequence.

5. Gel electrophoresis: The PCR products are separated and visualized using agarose gel electrophoresis. If the EWS/FLI1 fusion transcript is present, a specific-sized band should be observed on the gel. Absence of this band indicates that the fusion transcript is not present.

It is important to note that the primer design and optimization of PCR conditions are critical to ensure specificity and sensitivity. Multiple controls, including known positive samples and negative controls, should be included in the experiment.

Overall, RT-PCR with primers designed to yield a product only if the EWS/FLI1 translocation has occurred is a valuable molecular technique to detect this fusion transcript in Ewing’s cancer, aiding in diagnosis and subsequent treatment planning.

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