Quantification of RNA degradation by semi-quantitative duplex and competitive RT-PCR: a possible indicator of the age of bloodstains?

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Abstract

In vitro RNA degradation is a complex and non-linear process which can serve as indicator for the quality and age of stains. We have developed a semi-quantitative duplex reverse transcription-polymerase chain reaction (RT-PCR) assay which, in combination with competitive RT-PCR using an external standard, allows quantification of RNA degradation levels. Using this method, we have investigated 106 bloodstains stored up to 15 years. The distribution of the peak area quotients of standard and target messenger-RNA (mRNA) as measured by laser-induced fluorescence capillary electrophoresis was closely correlated with the age of the samples. Further statistical analysis showed that bloodstains with age differences of 5 years and more exhibit statistical significant variances in peak area quotients of both housekeeping genes included in this study, β-actin and cyclophilin. This can be of value when evidence from old cases is re-investigated. Our data show, that, although RNA continues to be degraded in dried bloodstains, mRNA suitable for RT-PCR can be isolated from samples stored for at least 15 years.

Introduction

The estimation of the age of dried bloodstains is a largely unsolved problem in forensic science. Several methods such as HPLC analysis of “decomposition peaks”, analysis of visible absorption spectra or remission analysis have been suggested for this purpose but never where established as routine technique in forensic casework [1], [2], [3], [4], [5], [6], [7], [8]. With the introduction of molecular technologies into forensic science, open questions such as the determination of the origin of dried stains could be addressed using polymerase chain reaction. For example, the detection of cell-specific gene expression by reverse transcription-polymerase chain reaction (RT-PCR) recently was shown to be a suitable method for identification of menstrual blood and spermatozoa [9], [10], [11].

Since forensic stains frequently are exposed to different environmental conditions for months or even years before they are collected and transferred to the laboratory, the question of nucleic acid stability under these conditions is of crucial importance. Whereas DNA seems to be stable in dried stains as long as no bacterial overgrowth occurs, RNA is believed to be rapidly degraded in vitro. However, even if stored under appropriate conditions, DNA also undergoes further fragmentation requiring more sensitive detection methods when stains generated years or decades ago are investigated. On the other hand, it could be shown that RNA isolation and analysis is possible from dried bloodstains kept for several years and from archival tissue materials [12], [13]. Messenger-RNA (mRNA) suitable for RT-PCR could be isolated from dried bloodstains after up to 6 months of storage as shown by the authors [9], [10].

The discrepancy between these findings and the widespread believe in the general instability of RNA has prompted us to investigate systematically the degree of RNA degradation in dried stains stored for longer time periods. The objective of this study was two-fold: to show that RNA suitable for RT-PCR can be extracted from bloodstains stored for several years and that the degradation level can be used as indicator of the age of bloodstains. For this purpose, it was necessary to quantify RNA with a reliable and reproducible technique. Recent studies have described several methods for quantification of RNA degradation based on competitive RT-PCR [14], [15], [16], but none of them was designed for use with RNA extracted from dried bloodstains. Therefore we had to develop and evaluate quantitative RT-PCR assays adjusted to small RNA amounts. We included two different approaches in this study, semi-quantitative duplex RT-PCR using an internal standard and competitive RT-PCR using a modified mRNA as external standard. Here, we present these techniques together with results from investigations on dried bloodstains stored for up to 15 years.

Section snippets

Sample preparation

Bloodstains were prepared using venous blood samples in EDTA-free tubes taken from healthy volunteers who had signed informed consent. In addition, artificial stains made for previous studies [9], [10], were re-used. Aliquots of 10 μl blood were pipetted onto clean cotton fabric and allowed to air-dry. The samples were stored for up to 5 years at room temperature protected from sunlight. A total of 90 stains from 41 different individuals were included in the study plus 16 samples which were left

Results

Amplification curves obtained from β-actin- and cyclophilin-specific PCR with aliquots of a single sample (storage time 0 day) amplified for 16–36 cycles show that the phase with exponential amplification of both fragments ranged between 20 and 26 cycles for β-actin (Fig. 3a) and between 26 and 31 cycles for cyclophilin (Fig. 3b). Whereas qPAcyc was constant over all cycles indicating an equal amplification efficiency of the endogenous and the modified fragment, qPAact decreased between the

Discussion

With the development of RNA-based diagnostic techniques in clinical and forensic medicine, in vitro RNA degradation has attracted increasing attention of scientists involved in RNA research. RNA was shown to be stable for several weeks in dried blood and serum spots which are used in virology [18], [19], [20], [21] and human genetics [22], [23], [24] for diagnostic purposes because storage, transport and analysis are facilitated especially under unfavorable conditions. Air-dried, unstained

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