An evidence based strategy for normalization of quantitative PCR data from miRNA expression analysis in forensically relevant body fluids

https://doi.org/10.1016/j.fsigen.2014.03.011Get rights and content

Abstract

Micro-RNA (miRNA) based analysis of body fluids and composition of complex crime stains has recently been introduced as a potential and powerful tool to forensic genetics. Analysis of miRNA has several advantages over mRNA but reliable miRNA detection and quantification using quantitative PCR requires a solid and forensically relevant normalization strategy.

In our study we evaluated a panel of 13 carefully selected reference genes for their suitability as endogenous controls in miRNA qPCR normalization in forensically relevant settings. We analyzed assay performances and variances in venous blood, saliva, semen, menstrual blood, and vaginal secretion and mixtures thereof integrating highly standardized protocols with contemporary methodologies and included several well established computational algorithms.

Based on these empirical results, we recommend normalization to the group of SNORD24, SNORD38B, and SNORD43 as this signature exhibits the most stable expression levels and the least expected variation among the evaluated candidate reference genes in the given set of forensically relevant body fluids.

To account for the lack of consensus on how best to perform and interpret quantitative PCR experiments, our study's documentation is compliant to MIQE guidelines, defining the “minimum information for publication of quantitative real-time PCR experiments”.

Introduction

RNA based analytical methods are on the rise in forensic molecular biology [1] and early international trial exercises for forensic RNA analysis have already been conducted [2], [3], [4]. The analysis of differential expression of mRNA may be used in forensic settings to identify body fluid components of mixed stains [5], to estimate wound or stain age [6], [7], detect pregnancy [8], and to help discern the cause of death [9].

There are, however, drawbacks associated with the analysis of mRNA, e.g. its susceptibility to degradation and lack of specificity in the identification or discrimination of particular body fluids especially vaginal secretions [10], [11]. Therefore, in addition, feasibility and practicability of forensic miRNA analysis [12] based on quantitative PCR (qPCR) is being assayed since recently by several groups [13], [14], [15].

Quantitative PCR is widely considered as the gold standard for the quantification of miRNA expression but for qPCR to deliver a reliable and biologically meaningful report of target molecule numbers an accurate and relevant normalization of non biological variances is essential [16], [17], [18], [19]. A robust normalization strategy that is specific for a particular experimental setup should encompass an individual and evidence based selection of one or a group of reference genes [20], [21], [22]. Therefore, in the present study, we present a group of endogenous reference genes selected on the base of empirical evidence for the normalization of qPCR data from expression analysis of 13 preselected miRNAs in forensically relevant body fluids.

Section snippets

Adherence to the MIQE guidelines

To facilitate reliable and unequivocal interpretation of the qPCR results reported herein, all information that is rated ‘essential’ according to the MIQE guidelines [23] is reported, where applicable.

Samples

Samples for each tested body fluid, i.e. venous blood, saliva, vaginal fluid, menstrual blood, and semen, were collected from healthy volunteers, after obtaining informed consent.

Venous blood was collected by venipuncture using dry vacutainer tubes and spotted onto sterile cotton swabs. For

Results

Quantity and integrity of total RNA varied notably among samples of the same body fluid as well as between groups with RIN values generally ≤4 (Table 1). Overall, saliva samples exhibited the lowest (total RNA concentration: 3.6–25.3 ng/μl; RIN: n.d.–1.8) and vaginal secretion samples the highest overall values (total RNA concentration: 41.9–257 ng/μl; RIN: 2.9–4).

RT(−)-controls for RNU6-1 and SNORD48 showed scarce unspecific amplification and these markers were excluded from further analyses

Discussion

For qPCR to deliver reliable and biologically meaningful results an accurate and relevant normalization of non biological variances is essential [16], [17], [18], [19]. Non-biological variances can include variations in PCR efficiency, amount of starting material by sample-to-sample variation, RNA integrity, RT efficiency and cDNA sample loading [34], [35], [36]. This has to be accounted for especially when, as in most forensic settings, samples have been obtained from different individuals,

Conclusion

Herein we analyzed 13 potential reference genes and empirically determined SNORD24, SNORD38B and SNORD43 to be the most stable endogenous reference genes for a reliable normalization of qPCR data from forensic miRNA expression analysis of body fluids in a set of body fluid samples.

Acknowledgements

We would like to thank all volunteers for their kind donation of sample material. We also thank the Deutsche Forschungsgemeinschaft (DFG) CO 992/3-1 for funding this project.

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