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7. For RNA extraction method B, resuspend cells and transfer a maximum of 2 x lo7 mto a microfuge tube. After pelleting the cells, remove PBS, dislodge the pellet by vortexing briefly, add 500 pL GTC (without CsCl) and vortex for 10 s If < 1O6 cells, add 5 pg E coli tRNA 8 Store GTC lysates at 4°C for up to 1 wk, or long-term at -2O’C. 2. 1. Method A (Modified from ref. 15) 1. Put 1 mL CsCl cushion into a Beckman (Fullerton, CA) conical ultracentrifuge tube and layer the GTC lysate carefully on top.
4 at 0°C with 2M HCL (do not autoclave). 2 Phosphate-buffered sahne (PBS): Made up from stock tablets (Oxold, Basmgstoke, UK) and autoclaved. 3. 0: Neutralize 1M trisodmm citrate with 1M citric acid. 4 GTC: 4M Guanidmmm thlocyanate (Fluka, Buchs, Switzerland), 5 mM EDTA, 25 mM citrate, pH 7 0, 0 5% sarcosyl (BDH, Poole, UK). 1 pL P-mercaptoethanol/mL of GTC m-mediately before use. 5. 0. Treat with DEPC (see Note 1). 6. 0. 7. 0, with glacial acetic acid and treated with DEPC Detection of BCR-ABL 27 8 70% ethanol made up with DEPC-treated water.
Products were resolved by 8% nondenaturing PAGE and electrophoretically transferred to nylon membranes. The second band observed in the dilution lanes is artifactual and does not compromise residual disease assessment. Dot blots provide a useful first screening method but do not give confident detection of low-level MRD. At times of polyclonal lymphocytosis seenin remission samplesafter the end of treatment or BMT, nonspecific hybridization of the probe to the products generated by this expanded normal population can lead to false positive MRD detection (20).