SEROLOGY BY qPCR ARRAY

A qPCR array was designed to facilitate forensic serological identification of unknown stains and fluids. In our approach, the flow-through of a DNA purification column is further purified for total RNA. Therefore, the DNA preparation is fully available for forensic DNA analysis whereas the RNA fraction is processed for serological examination by mRNA analysis. The RNA fraction is first subjected to DNase treatment to remove contaminating DNA and then reverse-transcribed to generate cDNA which is subsequently assayed by real-time (qPCR). The cDNA preparation is simply added to a qPCR master mix and dispensed into a qPCR array containing 32 separate assays, each of which contains primers and probe for a specific gene transcript. The transcripts queried in the array represent candidate gene markers for semen, seminal fluid, saliva, blood, menstrual blood, vaginal secretions, skin, sweat, and urine. The specimen types evaluated included all of the above except for sweat. In addition, up to six assays for housekeeping genes were included in the array to normalize the quantity of RNA extracted. The qPCR arrays were pre-loaded in triplicate on a 96-well plate therefore up to three different cDNA preparations may be simultaneously tested per plate or a single cDNA preparation may be tested in triplicate. Sample loading is accomplished in minutes and qPCR run time is completed in approximately 50 minutes. Following qPCR, amplification plots, Ct values, and ΔΔCt values are analyzed.