Preparation of Agarose gel
Generally, two different type of agarose are used in Single cell gel electrophoresis assay for blood and tissue/organ samples.
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1)
Low Melting Agarose (LMA) - 1% LMA is mixed into the Phosphate buffered saline (PBS) and heat until the agarose dissolve in PBS. LMA stored at 4 °C until required. When needed, briefly melt agarose in microwave and kept on dry/water bath maintained at 37 °C (Shukla et al. 2011).
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2)
Normal melting agarose (NMA) - 1% NMA is mixed into the distilled water and heat until the agarose dissolves in distilled water. It always freshly prepared and kept on dry bath at 60 °C during the preparation of base slides (Shukla et al. 2011).
For semen sample, two different types of high resolution agarose gel are prepared which can be used for SCGE assay
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1)
0.7% Agarose- 70 mg of 3:1 high resolution agarose mixed with 9 ml of distilled water in a 100 ml glass beaker and microwaved until clear solution formed. Then add 1 ml of PBS in this clear solution and microwave the solution again. Keep this solution on dry bath and maintain the temperature at 55 °C for the experiment (Simon et al. 2013).
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2)
1% Agarose- 1% agarose is also prepared with similar manner as preparation of 0.7% Agarose and finally kept on dry bath at 55 °C for the experiment (Simon et al. 2013).
Preparation of base slide
For blood and tissue/organ sample
End frosted microscopic slides are used for the SCGE assay. Slides are first dipped in methanol and burnt over a blue flame to remove machine oil and dust. Thereafter, slides are vertically dipped in 1% NMA gel up to two-third of the frosted area (the frosted end prevents the gel from slipping off the slide). Wipe the underside of slide to remove agarose and lay the slide in a tray or on a flat surface to dry. The slides may be air dried or warmed at 50 °C for quicker drying. The slides are stored in a dry slide box at room temperature until needed, and are generally prepared the day before use. (NOTE: Slides should be labeled at the frosted end with pencil before storage to avoid confusion of the side with/without gel) (Bajpayee et al. 2005; Shukla et al. 2014).
For semen sample
In case of semen sample, slide is pre-coated with 0.7% agarose instead of NMA. Thereafter, a microgel layer of agarose is prepared prior to put cells on the 0.7% agarose pre-coated slide. In this step, 200 μl of 1% agarose is placed on the pre-coated slide and covered with cover slip. Agarose is allowed to solidify at room temperature for 5 min (or 1 min, if on ice). Before adding cell suspension onto the slide, cover slip is gently removed (Simon et al. 2013).
Sample preparation
Blood- 50 μl of blood obtained from the deceased body/crime scene to perform this assay. This sample is first diluted with 50 μl PBS and then mixed with equal volume of 1% LMA and proceed for the experiment (Bajpayee et al. 2005).
Sperm- Spermatozoa cells are extracted from dried cloths/crime scene by soaking for 5 min at 37 °C and then subsequent washings with 200 μl PBS. Thereafter, spermatozoa cells are collected through centrifuged and re-suspended in 1 ml PBS. This suspension is then mixed with 0.7% agarose and proceeds for the experiment (Simon et al. 2013).
Solid Organs/tissues- A small piece of blood rich organ (eg Spleen, liver) placed in freshly prepared chilled mincing solution (PBS, with 20 mM EDTA and 10% DMSO) and chopped into pieces with a pair of scissors. The pieces are allowed to settle and the supernatant containing the single cells is mixed with 1% LMA and preceded further for the experiment (Shukla et al. 2016).
Slide preparation
For blood and tissue/organ sample
Slide preparation is done according to a modified method of Tice et al. (2000). Cells embedded in Low melting agarose (LMA) are layered on normal melting agarose (NMA) pre-coated base slides. A cover-slip is placed on the slide to ensure that the gel is evenly spread, and the slide is kept on ice to allow the gel to solidify. A third layer of 90 μl LMA (0.5%) is added and again allowed to solidify on ice. Duplicate slides are prepared for each sample (Bajpayee et al. 2005).
For semen sample
Cell embedded in the agarose (50 μl of 0.7% agarose is mixed with 0.5 μl of fresh semen) first layered onto the pre-coated slide and covered with cover slip. The slides are left at room temperature to solidify the gel for 5 min (or 1 min, if on ice), then cover slip is gently removed and 200 μl of 0.7% agarose is added on each slide and covered with cover slip. Again the slide is kept on ice to solidify the gel and the cover slip is finally removed to proceed for lysis (Simon et al. 2013).
Lysis
This step helps to lyse the cells using lysing solution with detergent. Presence of high salt in lysing solution helps to remove cell membranes, bulk of proteins, cytoplasm and nucleoplasm. It further disrupts nucleosomes and almost all histones being solubilized by the high salt. Furthermore, it form nucleoids consisting of a nuclear matrix composed of ribonucleic acid (RNA), proteins and containing negatively charged super coiled loops of DNA (Collins 2004; Shukla et al. 2011). In this step cover-slips are removed and the slides immersed in a freshly prepared and chilled lysis solution (146.1 g NaCl, 37.2 g EDTA, 1.2 g Tris, pH 10) with 1% Triton X-100 and 10% Dimethyl sulfoxide (DMSO) added just before use. The slides are kept in lysis solution overnight at 4 °C (Bajpayee et al. 2005; Shukla et al. 2014).
NOTE: The purpose of the DMSO in the lysing solution is to scavenge radicals generated by the iron released from hemoglobin when blood or tissues are used.
In case of semen sample, 0.5 mg/ml of proteinase K (PK needs to be incubated at 37 °C to inactivate DNAse impurities which may induce additional DNA damage) and 2 mg/ml reduced glutathione (antioxidant) should be added into the fresh to pre-warmed (37 °C) lysing solution. In case of semen sample despite of Triton X 100, Sodium lauryl sulphate (SLS) can be used as detergent (Simon et al. 2013).
DNA unwinding
With the help of this step, any breaks present in the DNA cause the super coiling to relax locally and loops of DNA are then free. This step proceeds after lysis of the cells embedded on base slide. In this step, the slides are placed first in a horizontal gel electrophoresis tank and then tank pour with fresh and chilled electrophoresis solution (1 mM EDTA and 300 mM NaOH, pH > 13). The slides are left in this solution for DNA unwinding for 25 min.
Electrophoresis
In case of blood and tissue samples, electrophoresis is performed at high pH (> 13) which facilitates denaturation of DNA (because of the disruption of hydrogen bonds between double-stranded DNA) and expression of alkali labile sites as frank breaks. The electrophoresis would pull the damaged DNA towards the anode thus making the distinct ‘tail’ of the comet. Electrophoresis is conducted at 4 °C with constant voltage 0.7 V/cm for blood (Bajpayee et al. 2005) and organs/tissue for 30 min. All steps are performed under dimmed light (Shukla et al. 2014).
In case of semen sample electrophoresis is performed at pH 10 and electrophoresis solution contain 0.5 M NaCl, 1 mM EDTA, 0.1 M tris base and 0.2% DMSO (Simon et al. 2013).
Neutralization and staining
After electrophoresis, the excess alkali is neutralized with Tris buffer (0.4 M, pH 7.5). When neutralization step occur, extruded DNA (single and double strand breaks) which are migrate towards anode, formed hair pin loop like structure. And when it stained with DNA binding dye, dye molecules are intercalate in between loop of extruded DNA and become visualized under fluorescent microscope. In the staining, slides are stained with any one of the DNA binding dye (Ethidium Bromide, Propidium Iodide, DAPI or YOYO) and stored in a humidified slide box until scored (Paul and Bhattacharya 2012). Ethidium Bromide is most commonly used DNA binding dye in the Comet assay.
Scoring of slides
Slides are scored using an image analysis system (Andor Technology Belfast, U.K.) attached to a microscope equipped with fluorescence attachment and appropriate filter (N2.1 excitation wavelength 515–560 nm - emission wavelength 590 nm). The microscope is connected to a computer through a charge coupled device (CCD) camera to transport images to software (Komet 5.5) for analysis. Generally, 100 randomly selected cells are analyzed per sample. The comet parameters recorded are Olive tail moment (OTM, arbitrary units), tail DNA (%) and tail length (migration of the DNA from the nucleus, μm; TL) as shown in Fig. 2.
These three parameters of SCGE assay are defined as:
Tail Moment: The product of the tail length and % of total DNA present in the tail of migrated DNA is known as Tail moment.
Tail Moment = Tail Length x % DNA Tail
Tail Length: Tail Length is defined as the length of DNA migration from the body of the nuclear core. It is used to assess the degree of DNA damage.
% Tail DNA: % Tail DNA is the fraction of total DNA present in tail of migrated DNA.