mRNA profiling for body fluid identification by reverse transcription endpoint PCR and realtime PCR

Haas, Cordula; Klesser, B.; Maake, Caroline; Bär, W.; Krätzer, A.

doi:10.1016/j.fsigen.2008.11.003

Cited by 244 publications

(152 citation statements)

References 25 publications

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“…However, currently no housekeeping gene is described that is suitable for the detection of all tested body fluids. Saliva and semen, for example, show very little or no expression of common housekeeping genes (GAPDH, 18S rRNA, TEF, UCE), presumably because of limited cell metabolism in spermatozoids and the desquamated cells of the cheek mucosa [14,17]. Additionally, the high abundance of housekeeping genes relative to mRNA may result in the titration of critical PCR components thereby affecting the overall sensitivity of the multiplex system.…”

Section: Discussionmentioning

confidence: 99%

“…organic extraction) as described previously [6] or with column-based kits (RNeasy Mini Kit or AllPrep DNA/RNA Mini Kit, Qiagen, Hombrechtikon, Switzerland and Germantown, MD), according to the manufacturer's protocol, with some adaptations [14]. Casework and environmentally compromised bloodstains were incubated at 56 o C for up to three 7/38 hours in RLTplus buffer for improved extraction and recovery of RNA from challenging samples.…”

Section: Rna Extractionmentioning

confidence: 99%

“…Primers for the amplification of gene specific sequences were obtained from published sources [6,13,14,16]. Additional primer sets were designed for HBA, ANK1 and CD3G, using the Primer3 Software (Electronic supplementary data, Table S1).…”

Section: /38mentioning

confidence: 99%

“…A number of mRNA markers have been identified for the forensically most relevant body fluids: blood, saliva, semen, vaginal secretions and menstrual blood [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17]. RNA is notorious for its rapid post-mortem and in vitro decay, but several reports have pointed out an unexpectedly high stability of RNA in forensic stains [18][19][20].…”

Section: Introductionmentioning

confidence: 99%

“…Blood was chosen for its easy availability, stability and the number of reported markers [6][7][8][9][10][13][14][16][17][19][20]. The following blood markers were included in the study: HBA and HBB are the alpha-and betasubunits of hemoglobin A [21,22]; aminolevulinate synthase 2 (ALAS2) is an erythroid-specific mitochondrial enzyme, that catalyzes the first step in the heme biosynthetic pathway [23]; CD3 gamma molecule (CD3G) is part of the T-cell receptor-CD3 complex, which plays an important role in coupling antigen recognition to several intracellular signal-transduction pathways [24]; ankyrin 1 (ANK1) is an erythrocyte membrane protein, which provides the primary linkage between the membrane skeleton and the plasma membrane [25]; the erythroid form of porphobilinogen deaminase (PBGD) is the third enzyme of the heme biosynthetic pathway [26]; β-spectrin (SPTB) is an erythrocyte membrane protein [27]; Aquaporin (AQP9) is a water channel protein expressed in peripheral leukocytes [28].…”

Section: Introductionmentioning

confidence: 99%

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Selection of highly specific and sensitive mRNA biomarkers for the identification of blood

Haas

Hanson

Krätzer

et al. 2011

Forensic Science International: Genetics

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In the present work, we have evaluated eight reportedly blood-specific mRNA markers (HBB, HBA, ALAS2, CD3G, ANK1, PBGD, SPTB and AQP9) in an attempt to determine the most suitable ones for use in forensic applications based on their sensitivities, specificities and performance with casework samples.While varying levels of expression were observed, all markers were relatively sensitive requiring as little as 1 ng of RNA input into the reverse transcription (RT) reaction. In singleplex reactions, seven of the eight analyzed blood markers (all except AQP9) demonstrated a high degree of specificity for blood. In multiplex reactions, non-reproducible cross-reactivity was observed for several of the mRNA markers, which was reduced and, in most cases, eliminated when less input total RNA was used. Additionally, some cross-reactivity was observed with tissue and animal samples. Despite differences in the observed sensitivity and specificity of the blood markers examined in this study, a number of the candidates appear to be suitable for inclusion in appropriately validated multiplex mRNA-based body fluid identification systems.

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Section: Discussionmentioning

confidence: 99%

Section: Rna Extractionmentioning

confidence: 99%

Section: /38mentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 3 more Smart Citations

Selection of highly specific and sensitive mRNA biomarkers for the identification of blood

Haas

Hanson

Krätzer

et al. 2011

Forensic Science International: Genetics

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DNA analysis: Current Practice and Problems

Court

2011

Current Practice in Forensic Medicine

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Microarray screening and qRT‐PCR evaluation of microRNA markers for forensic body fluid identification

Park

Kwon

et al. 2014

Electrophoresis

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MicroRNAs (miRNA) are a class of small (∼22 nucleotides) noncoding RNAs that regulate diverse biological processes at the post-transcriptional level. MiRNAs have great potential for forensic body fluid identification because they are expressed in a tissue specific manner and are less prone to degradation. Previous studies reported several miRNAs as body fluid specific, but there are few overlaps among them. Here, we used a genome-wide miRNA microarray containing over 1700 miRNAs to assay 20 body fluid samples and identify novel miRNAs useful for forensic body fluid identification. Based on Shannon Entropy and Q-statistics, 203 miRNAs specifically expressed in each body fluid were first selected. Eight miRNAs were then selected as novel forensically relevant miRNA markers: miR-484 and miR-182 for blood, miR-223 and miR-145 for saliva, miR-2392 and miR-3197 for semen, and miR-1260b and miR-654-5p for vaginal secretions. When the eight selected miRNAs were evaluated in 40 additional body fluid samples by qRT-PCR, they showed high sensitivity and specificity for the identification of the target body fluid. We suggest that the eight miRNAs may be candidates for developing an effective molecular assay for forensic body fluid identification.

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mRNA profiling for body fluid identification by reverse transcription endpoint PCR and realtime PCR

Cited by 244 publications

References 25 publications

Selection of highly specific and sensitive mRNA biomarkers for the identification of blood

Selection of highly specific and sensitive mRNA biomarkers for the identification of blood

DNA analysis: Current Practice and Problems

Microarray screening and qRT‐PCR evaluation of microRNA markers for forensic body fluid identification

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