HemaVision Multiplex RT-PCR System for Typing/Subtyping of Leukemia

HemaVision is a line of products offered by DNA Technology. We use their multiplex RT-PCR system for typing leukemia (cat number DNAT–HV1); this kit can screen and type 28 different translocations.

We use this tool for the screening of leukemic samples. Once screened and typed, we use independent real-time quantitative PCR kits for quantification. Very initially, we found the HemaVision procedure slightly burdensome because of the longer turnaround time (> 6 hrs), but after seeing the sensitivity, specificity, and reproducibility of the assay, we feel content with it.

The kit comes as two separate boxes. Box 1 contains sixteen (8 x 2) tubes (M1 – M8). These reagents are used for the screening of the sample. The volume in each vial is 155 ul. This can screen 25 patient samples. Box 2 contains 74 tubes (37 x 2) of "slit-out" PCR mix which are used for the typing of those samples which give a positive reaction with box 1. The volume of each tube is 95 ul. This can type 15 patient samples. Additional kits and reagents for total RNA extraction, cDNA synthesis kit and amplification (Taq polymerase and buffers) need to be purchased separately.

To start with, we use Qiagen's Blood RNA kit (recommended by manufacturers) for extracting total RNA and Takara's 1st strand cDNA synthesis kit. HemaVision requires a large quantity of cDNA: approx 50 ul/sample (if RNA concentration is around 80 ng/ul). The reason so much cDNA is required is because every sample has to undergo 4 rounds of PCR: twice for the master PCR (box 1) and again twice during the split-out PCR (box 2), if the sample is positive.

Once we have our cDNA, we set up the first master PCR amplification which consists of 8 parallel PCR reactions for each patient sample. Here each PCR reaction (M1 – M8) contains a different primer solution specific for the amplification of a different translocation. As previously described, the first master PCR must be performed twice with separate vials. We check the amplification product after the second PCR. If we get a positive result in any of the 8 tubes (i.e. amplification has occurred), we perform the split-out PCR (or otherwise report that the sample is negative). The presence of a specific product in one of the 8 lanes from one sample determines which of the corresponding set of split-out primer solutions must be used in the split-out PCR amplification. For example, a specific band in lane 2 means that the Split-out Primer Solution set denoted M2A - M2E must be used. Finally, we inspect the amplified product of the nested split-out PCR on gel and report the translocation accordingly.