Determination of an Effective Housekeeping Gene for the Quantification of mRNA for Forensic Applications*

Moreno, Lilliana I.; Tate, Courtney M.; Knott, Erika L.; McDaniel, Jade E.; Rogers, Stephanie S.; Koons, Barbara W.; Kavlick, Mark F.; Craig, Rhonda L.; Robertson, James

doi:10.1111/j.1556-4029.2012.02086.x

Cited by 22 publications

(17 citation statements)

References 47 publications

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“…Two of the housekeeping genes tested in this study, B2M and UBC, proved to be quite sensitive and were detected in most body fluid stains (Table S8). Moreno et al tested the expression levels of a selection of housekeeping genes and showed B2M to be expressed most consistent among body fluids [48]. Based on our data, these housekeeping genes do not appear to be particularly good positive controls in the sense that they should reliably show positive results when biological material is present.…”

Section: Discussionmentioning

confidence: 65%

RNA/DNA co-analysis from human menstrual blood and vaginal secretion stains: Results of a fourth and fifth collaborative EDNAP exercise

Haas

Hanson

Anjos

et al. 2014

Forensic Science International: Genetics

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The European DNA Profiling Group (EDNAP) organized a fourth and fifth collaborative exercise on RNA/ DNA co-analysis for body fluid identification and STR profiling. The task was to identify dried menstrual blood and vaginal secretion stains using specific RNA biomarkers, and additionally test 3 housekeeping genes for their suitability as reference genes. Six menstrual blood and six vaginal secretion stains, two dilution series (1/4-1/64 pieces of a menstrual blood/vaginal swab) and, optionally, bona fide or mock casework samples of human or non-human origin were analyzed by 24 participating laboratories, using RNA extraction or RNA/DNA co-extraction methods. Two novel menstrual blood mRNA multiplexes were used: MMP triplex (MMP7, MMP10, MMP11) and MB triplex (MSX1, LEFTY2, SFRP4) in conjunction with a housekeeping gene triplex (B2M, UBC, UCE). Two novel mRNA multiplexes and a HBD1 singleplex were

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Section: Discussionmentioning

confidence: 65%

RNA/DNA co-analysis from human menstrual blood and vaginal secretion stains: Results of a fourth and fifth collaborative EDNAP exercise

Haas

Hanson

Anjos

et al. 2014

Forensic Science International: Genetics

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“…Anderson et al previously reported that rRNA 18S was more stable than mRNA ACTB in the same species, 27) while Moreno et al recently showed that beta 2 microglobulin (B2M) was equal to ACTB as a useful housekeeping gene in forensic samples. 28) For this reason we compared the stability of ACTB in saliva, semen, and blood stains at various temperatures, and found that relative Ct values in all body ‰uid stains were signiˆcantly higher at 4°C in the refrigerator. Moreover, relative Ct values increased more in all body ‰uid stains at room temperature (22 25°C) after 6 months than at 4°C or 35°C.…”

Section: Discussionmentioning

confidence: 99%

Identification of Body Fluid Stains Using Real-time RT-PCR: Discrimination Between Salivary, Nasal, and Vaginal Secretions

Sakurada

Akutsu

Watanabe

et al. 2013

Jpn. J. Forensic. Sci. Tech.

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In a criminal investigation, it is important to clarify the origin of the extracted DNA from available biological samples. In recent years, a new approach for the identiˆcation of body ‰uid stains has been demonstrated and involves a comparison of speciˆc mRNA expression levels. Here, we used real-time RT-PCR to examine the expression levels of STATH, HTN3, MUC4, and ESR1 genes from salivary, nasal, and vaginal secretions which are often thought to be di‹cult to discriminate between. STATH was expressed at high levels in all salivary (dCt＝1.27±3.18, n＝ 10) and all nasal secretions (dCt＝-0.99±3.94, n＝8), and at low levels in 3 of the 10 vaginal secretions (dCt＝16.34-17.47). HTN3 was expressed at high levels in all salivary secretions (dCt＝0.67±3.08). MUC4 was expressed at relatively high levels in all vaginal secretions (dCt＝4.97±2.36, n＝10), and at moderate levels in 5 of the 10 salivary secretions (dCt＝8.55-10.40) and in 4 of the 8 nasal secretions (dCt ＝6.56-8.23). ESR1 was expressed at relatively high levels in all vaginal secretions (dCt＝5.79±2.90), but no expression was found in salivary and nasal samples. These results indicate the possibility for speciˆc discrimination between various body ‰uids. To test the practicality of this, gene sensitivity and stability were examined in saliva, semen, and blood stains. Several genes, including ACTB, STATH, HTN3, PRM2, SEMG1, and HBB, were shown to be detectable even in small volume (0.1 mL) stains and were also expressed in 1-year-old stains. mRNA stability was dependent on favorable environmental factors, such as dryness and shading. In addition, the successful detection of target genes from mixed stains and simulated casework samples might promise the utility of this assay. The mRNA assay therefore appears to be a novel tool for body ‰uid identiˆcation from biological samples.

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“…Due to its conceptual simplicity, sensitivity, specificity, and speed, qPCR applications can be found in a variety of fields, including medicine and the life sciences [1, 2]. In clinical diagnostics, qPCR is broadly used for the detection and quantification of bacterial and viral loads, gene dosage determination, cancer diagnostics, and applications in forensic medicine [3–7]. …”

Section: Introductionmentioning

confidence: 99%

GAPDH Pseudogenes and the Quantification of Feline Genomic DNA Equivalents

Helfer-Hungerbuehler

Widmer

Hofmann‐Lehmann

2013

Molecular Biology International

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Quantitative real-time PCR (qPCR) is broadly used to detect and quantify nucleic acid targets. In order to determine cell copy number and genome equivalents, a suitable reference gene that is present in a defined number in the genome is needed, preferably as a single copy gene. For most organisms, a variable number of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) pseudogenes have been reported. However, it has been suggested that a single-copy of the GAPDH pseudogene is present in the feline genome and that a GAPDH assay can therefore be used to quantify feline genomic DNA (gDNA). The aim of this study was to determine whether one or more GAPDH pseudogenes are present in the feline genome and to provide a suitable alternative qPCR system for the quantification of feline cell copy number and genome equivalents. Bioinformatics and sequencing results revealed that not just one but several closely related GAPDH-like sequences were present in the cat genome. We thus identified, developed, optimized, and validated an alternative reference gene assay using feline albumin (fALB). Our data emphasize the need for an alternative reference gene, apart from the GAPDH pseudogene, for the normalization of gDNA levels. We recommend using the fALB qPCR assay for future studies.

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Determination of an Effective Housekeeping Gene for the Quantification of mRNA for Forensic Applications*

Cited by 22 publications

References 47 publications

RNA/DNA co-analysis from human menstrual blood and vaginal secretion stains: Results of a fourth and fifth collaborative EDNAP exercise

RNA/DNA co-analysis from human menstrual blood and vaginal secretion stains: Results of a fourth and fifth collaborative EDNAP exercise

Identification of Body Fluid Stains Using Real-time RT-PCR: Discrimination Between Salivary, Nasal, and Vaginal Secretions

GAPDH Pseudogenes and the Quantification of Feline Genomic DNA Equivalents

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