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miRNA Profiling to Distinguish Bodily FluidsRachel Markert
Micro-RNA Background• Small noncoding RNA• Used in cellular processes, posttranslational• miRNA regulation used for controlling gene expression. • Binds to mRNA
(http://cnx.org/content/m36053/latest/mirna.png)
Experiment: Step 1• Fluids were obtained through buccal swabs for saliva and vacutainer tubes for blood.• The microarray method used fluids from 4 females and 1 male• Verification by PCR used samples from 3 females and 2 males• Aged blood was made from one sample over one year
Experiment: Step 2• Extraction of RNA from all samples• Enriched for miRNAs with kits• Buffer used to remove erythrocytes from blood• RNA concentration was found using RNA integrity number
(RIN) from fluorometer and bioanalyzer
Experiment: Step 3• Microarrays were used • Probes were created as
reverse complements of miRNAs from known human sequences• Positive results for blood
and saliva were selected for from respective arrays
Experiment: Step 5• Mathematical equations were carried out for hierarchical
clustering
Var(t) is sample variance Var (t,i) is variance in group i, calculated already
Normalization for qPCR
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Courts et al. (2011)
Courts et al. (2011)
Courts et al. (2011)
FIG. 5—Expression of candidate miRNAs in different kinds of tissue.Upper panel: Normalized median expression of the three blood-miRNAs(miR-126, miR-150, miR-451) in blood, saliva, liver, muscle and brainLower panel: Normalized median expression of the three saliva-miRNAs(miR-200c, miR-203, miR-205) in blood, saliva, liver, muscle and brain.
FIG. 4—qPCR validation of candidate miRNAs for blood and saliva.Upper panel: relative expression of blood candidate miRNAs in blood comparedto saliva, for which expression has been arbitrarily set to 0; Centralpanel: relative expression of blood candidate miRNAs in aged blood comparedto saliva for which expression has been arbitrarily set to 0; Lowerpanel: relative expression of saliva candidate miRNAs in saliva comparedto blood for which expression has been arbitrarily set to 0.
Results• As seen previously, 3 candidates from both blood and saliva
contained specific miRNAs • Aged blood also resulted in miRNA assay, proving that miRNA
could withstand some degradation• A mixed sample of blood and saliva resulted in distinguishing
between the two types of samples
Future Applications• Hopefully, widespread use in forensic labs• Standardization of the procedure for validation and optimal
processes for distinction• More research into other body fluids, such as semen, vaginal
secretions, and menstrual blood• Use especially in rape cases
References• Courts, C; Madea, B. “Specific micro-RNA signatures for the
detection of saliva and blood in forensic body-fluid identification.” J Forensic Sci. v.56 no. 6 p. 1464-1470. 2011.
• Landgraf , P; Rusu , M; Sheridan , R; et al. “A mammalian microRNA expression atlas based on small RNA library sequencing.” Cell. v. 129 no. 7 p. 1401–14. 2007.