Sample isolation

Body fluids are complex by nature. They contain a range of components including enzymes, proteins and cellular debris that can interfere with BFID analysis. Therefore it is important, that an appropriate isolation method is selected. This need is exemplified by the

71 fact that many body fluids collected at a crime scene are limited and or of poor quality. To further complicate matters the source of the body fluid may be unknown, especially if found in trace amounts at a crime scene.

Therefore part of this research was focused on finding a universal isolation method for body fluids. Four different isolation methods were identified. All isolations were performed without specialist treatment towards specific body fluids (e.g. DTT treatment for semen).

2.3.1 Dynabeads

magnetic separation technology

One method explored was the Dynabeads® magnetic beads technology by Life Technologies. The technology is different from other commercial bead based kits (e.g. by Promega and Qiagen) as the uniform spherical nature of the sample allows for 360° binding of samples. Dynabeads are also paramagnetic or in other words are magnetised when a magnetic field is present. This means that the separation of genetic material is far gentler than with methods that use centrifugation. This is advantageous from the police and forensic practitioners perspective as there is a heavy backlog for processing casework samples.

Two different isolation kits were selected: the Dynabeads® mRNA DIRECT kit, which uses an oligo-dT tail for isolating polyadenylated mRNA and the Dynabeads® DNA DIRECT™ Universal kit, which uses a silica-like chemistry (Life Technologies, UK).

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2.3.1.1 Messenger RNA isolation using oligo-dT beads

The standard mRNA isolation protocol was used on all body fluids [238]. The oligo® (dT)25

beads were first prepared in this protocol. The beads are heavier than the solution and tend to gather at the bottom of the tube. Therefore homogenization is required before pipetting the solution into sterile 1.5 ml collection tubes (Fisher Scientific, UK). Note the beads come immersed in a pre-lysis/binding solution, 1 X PBS (pH 7.4).

The tube is then placed onto a DynaMag™-2 magnet (Life Technologies, UK) for 30 s to allow sufficient time for the beads and solution to separate. In most cases, separation of the beads occurs within the first 10 s of applying a magnet. A gelatinous looking streak then forms along the side of the tube wall allowing for the surrounding solution to be carefully removed, usually with smaller filtered pipette tips. The tube is then removed from the magnet to allow for the beads to separate. An additional lysis/binding buffer containing strong chaotropic agents is then added to the beads.

Preparation of the sample involved modification to the original protocol since it is designed for tissues and cultured cells. Samples collected using filter paper were prepared by cutting 1 cm (diameter) hole punches into collection tubes. Samples collected using cotton swabs were prepared by removing the entire swab head with sterile disposable scalpels (Swann Morton, UK). Samples were then treated with the same lysis/binding buffer and vortexed to break down the cell walls.

73 Samples then underwent mRNA extraction. All samples were transferred into the 1.5 ml tubes containing Dynabeads. Tubes were then rotated for 15 min. using an orbital shaker (VWR, UK) at room temperature to allow for hybridization between mRNA and oligo-dT beads to occur. Sample tubes were then placed on the magnet for two min to allow for the mRNA/Dynabeads complex to form. The supernatant was then carefully removed using a pipette. Samples were then washed four times with two different wash buffers, during which the mRNA/Dynabeads complex became more compact due to removal of impurities. Samples were then eluted using Tris-HCl (10 mM, pH 7.5).

2.3.1.2 DNA isolation using silica beads

The standard Direct DNA isolation protocol was used to isolate DNA [239]. The protocol was very similar to the mRNA protocol, except the Dynabeads came readily prepared in the lysis/binding buffer solution. The sample preparation protocol was similar to the sample preparation method developed for the mRNA protocol with exception of adding 1 X PBS to lyse and bind the cells.

Samples then underwent extraction by adding the Dynabeads to the solution in a single rapid pipetting action and incubated to allow for lysis and binding of the cells to occur. Samples were then separated with the Dynal magnet and allow for the DNA/Dynabeads complex to form. The lysis/binding solution was then removed through pipetting and then taken from the magnet. The sample was then washed twice with a single wash buffer to remove impurities. Samples were then eluted with a low buffer to reverse the binding conditions.

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2.3.2 Qiagen silica gel membrane technology

The Qiagen silica gel membrane technology was also explored as it is commonly used technique amongst forensic science providers. Two isolation kits were selected: RNeasy mini kit and QIAamp DNA blood mini kit (Qiagen, UK).

2.3.2.1 Total RNA isolation using silica columns

The “Purification of total RNA from animal cells using spin technology” protocol was used to isolate total RNA from body fluids [240]. This protocol can be divided into two steps: sample preparation and sample isolation. Minimal modifications were required for this protocol.

Samples collected using cotton swabs and filter paper were prepared in a similar manner as before, except a combination of high salt lysis/binding buffer and 70% ethanol were used to breakdown the cell walls.

Samples were then transferred to RNeasy® spin columns and centrifuged using a Hettich MIKRO temperature controlled centrifuge (VWR, UK). Flow through from the spin columns was discarded. Samples were then washed three times with two different wash buffers to remove impurities. Samples were then eluted in RNase-free water to reverse binding conditions in the silica bed.

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2.3.2.2 DNA isolation using silica columns

The “DNA purification from cotton swabs (spin protocol)” was used to isolate DNA [241]. This protocol has been divided into two steps: sample preparation and sample isolation. Minimal modification was required for this protocol.

Samples collected using cotton swabs and filter paper was prepared in a similar manner as before, except a combination of a high salt lysis/binding buffer, PBS (pH 7.5) and proteinase K were used to breakdown cell walls. Samples were then vortexed and incubated. Ethanol was then added to further promote binding.

Samples were then transferred into QIAamp mini spin columns and treated in the same manner as the RNeasy isolation method. They were then washed twice with two different wash buffers to remove contaminants and then eluted with a low salt elution buffer to remove the purified DNA sample from the silica bed.