Discover how Representational Difference Analysis of cDNA helps scientists identify critical mRNA expression differences between healthy and diseased cells
Imagine you have two nearly identical books, but one contains a few extra, critically dangerous sentences that the other lacks. Your mission is to find those specific sentences. This is the challenge faced by geneticists studying diseases like cancer .
The master blueprint containing all genetic information present in every cell.
Active photocopies of DNA blueprints that tell us what a cell is actually doing.
The functional molecules that carry out cellular activities based on mRNA instructions.
Extract mRNA from both cancer cells (Tester) and healthy cells (Driver), then convert to stable cDNA using reverse transcriptase .
Cut cDNA into fragments and attach different molecular "name tags" (adaptors) to Tester and Driver sequences.
Mix Tester cDNA with 100x excess Driver cDNA. Common sequences pair up, unique Tester sequences remain single-stranded.
Use PCR with primers specific to Tester adaptors to exponentially amplify only the unique, single-stranded Tester molecules.
Perform multiple rounds of subtraction and amplification to create highly purified difference products containing only unique genes.
| Gene Identifier | Function & Association | Role in Cancer |
|---|---|---|
| HER2/neu | Receptor signaling cell growth | Acts like "gas pedal stuck on" for uncontrolled division |
| VEGF | Vascular Endothelial Growth Factor | Promotes blood vessel growth to "feed" tumors |
| MMP-9 | Matrix Metalloproteinase-9 enzyme | Breaks down tissue allowing cancer spread |
The discovery of HER2/neu overexpression in breast cancers led directly to the development of Herceptin (Trastuzumab), a targeted therapy that specifically blocks the HER2 protein and has significantly improved survival rates for this aggressive cancer subtype .
| Reagent / Tool | Function in Experiment |
|---|---|
| Oligo(dT) Primer | Binds to poly-A tail of mRNA to initiate cDNA synthesis |
| Reverse Transcriptase | Copies mRNA template to synthesize complementary DNA |
| Restriction Enzymes | Molecular scissors cutting DNA at specific sequences |
| Adaptors | Short DNA strands providing PCR primer binding sites |
| DNA Ligase | Glue enzyme attaching adaptors to cDNA fragments |
| Taq DNA Polymerase | PCR workhorse enzyme replicating DNA strands |
| PCR Primers | Guide polymerase to amplify specific fragments |
While newer technologies like RNA sequencing now provide comprehensive transcriptome views, cDNA RDA remains a landmark technique in molecular biology history. It enabled systematic hunting for differentially expressed genes without prior knowledge, leading to countless discoveries in cancer, development, and immune response research.
Pioneered targeted gene discovery before high-throughput sequencing was available
Foundation for understanding gene expression patterns in disease pathology