Determination of an effective housekeeping gene for the quantification of mRNA for forensic applications.

Author(s): Moreno LI, Tate CM, Knott EL, McDaniel JE, Rogers SS, Koons BW, Kavlick MF, Craig RL, Robertson JM

Publication: J Forensic Sci, 2012, Vol. 57, Page 1051-8

PubMed ID: 22309221 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   The purpose of this study was to determine the effects of the type of bodily fluid and the assay type on the quantification of 6 housekeeping genes in air-dried specimens, with or without outdoor storage for 24 h. Blood, semen and saliva were spotted in equal volumes onto swabs and air-dried overnight, while vaginal secretions and menstrual blood were collected directly on swabs. A replicate set of specimens were left outside (average temp 25.5 degrees C, no precipitation) for 24 h.

   Summary of Findings:

   Amplification of GAPDH was successful from vaginal swabs, menstrual blood, and semen using conventional RT-PCR and a one-step real-time qRT-PCR producing a 226 bp amplicon, but CT values were below the standard curve when blood and semen were used with the one-step real-time qRT-PCR kit. Using a two-step real-time qRT-PCR kit with a FAM label and a 122 bp amplicon, GAPDH was amplified successfully from all specimens except 1 of 5 saliva specimens. Further, the two-step real-time RT-PCR kit with a FAM label had an average increase in GAPDH recovery of 96% over that observed with the one-step kit. After 24 h of storage outdoors, B2M, ACTB and PGK1 were the most highly expressed transcripts in saliva, blood, vaginal secretions, and menstrual blood, and PPIA, PGK1, and B2M were the most highly expressed transcripts in semen. Generally, GAPDH had the lowest expression across all bodily fluids, and ACTB and B2M were the most consistently expressed between bodily fluids. While interindividual variability in expression of all transcripts was low in blood, all transcripts showed high interindividual variability in semen, vaginal secretions and menstrual blood.

   Biospecimens

   • Bodily Fluid - Saliva
   • Bodily Fluid - Other
   • Bodily Fluid - Semen
   • Bodily Fluid - Blood

   Preservative Types

   • Other Preservative

   Diagnoses:

   • Normal

   Platform:

   Analyte	Technology Platform
   RNA	Real-time qRT-PCR
   RNA	RT-PCR

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Acquisition	Biospecimen location	Saliva Blood Menstrual blood Vaginal secretion Semen
   Storage	Storage duration	0 h 24 h
   Real-time qRT-PCR Specific	Targeted nucleic acid	B2M GAPDH ACTB PGK1 PPIA RPLP0
   Real-time qRT-PCR Specific	Technology platform	Two -step with FAM label One-step with TAMRA label Conventional PCR
   Real-time qRT-PCR Specific	Length of gene fragment	122 bp 226 bp

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