	US 7,588,921 B1
	Messenger RNA profiling: body fluid identification using multiplex real time-polymerase chain reaction (q-PCR)
John Ballantyne, Orlando, Fla. (US); and Jane Juusola, Glen Allen, Va. (US)
Assigned to University of Central Florida Research Foundation, Inc., Orlando, Fla. (US)
Filed on Sep. 21, 2005, as Appl. No. 11/232,277.
Claims priority of provisional application 60/612057, filed on Sep. 22, 2004.
Int. Cl. C12P 19/34 (2006.01); C07H 21/04 (2006.01)

U.S. Cl. 435—91.2 [536/24.3]

6 Claims

1. A method for identifying the presence of one or more body fluids from a human being in a single or mixed stain the fluids selected from the group consisting of non-menstrual blood, menstrual blood, saliva and semen, comprising the steps of:

a) obtaining a sample stain comprising a body fluid from a human being;

b) extracting total ribonucleic acid (RNA) from the sample stain;

c) treating the total RNA from step b) with an enzyme

d) initiating a reverse-transcriptase (RT) reaction by treating the total RNA from step c) with random decamers to produce cDNA;

e) amplifying a pair of body-fluid specific genes (BFGs) selected from the pairs: ALAS2 and SPTB, specific for non-menstrual blood, HTN3 and STATH, specific for saliva, PRM1 and PRM2, specific for semen, and MMP-7 and MMP-10, specific for menstrual blood, wherein said amplifying utilizes two primers and a probe for each BFG;

f) amplifying a housekeeping gene (HSK) selected from one of GAPDH and GNAS, wherein said amplifying utilizes two primers and a probe for each HSK;

g) determining the presence of the body fluid in the stain by measuring the cycle threshold (Ct) value-of the house keeping gene (HSK) and the Ct values of both BFGs, and then subtracting the Ct value of each BFG from the Ct value of the HSK gene, wherein a positive dCt (HSK-Ct minus BFG-Ct) value of each BFG indicates the presence of the corresponding body fluid in the stain.