Table 3.

Reagents and parameters tested for enrichment of exosomes.

Tested reagents and parameters*	Variations	Mean real-time data of miR-16 as Cq difference(CD) between the variants**
Type of enrichment reagent 1	MGP 1***	0
	MGP 2	1.48
	MGP 3	1.48
	MGP 4	1.60
	MGP 5	0.56
Volume of enrichment reagent 1	20 µl	0.25
	30 µl***	0
	40 µl	0.83
	50 µl	0.52
	60 µl	3.07
	90 µl	8.35
	100 µl	4.02
Incubation time using enrichment reagent 1	None***	0
	1 min	−0.03****
	10 min	0.20
	30 min	0.10
	60 min	1.13
Incubation temperature using enrichment reagent 1	4°C	0.23
	Room temperature***	0
	50°C	7.94
Incubation with enrichment reagent 1 on a Thermoshaker	No shaking***	0
	400 rpm	0.69
Type of enrichment reagent 2	Calcium chloride***	0
	Calcium acetate	0.51
	Ammonium chloride	1.64
	Zinc chloride	3.10
	Manganese (II) chloride	2.11
Volume of enrichment reagent 2	100 µl	0.11
	150 µl***	0
	300 µl	0.89
	450 µl	1.19
Incubation time using enrichment reagent 2	1 min	0.80
	3 min	0.45
	10 min***	0
	30 min	0.44
	60 min	0.62
Incubation temperature using enrichment reagent 2	4°C	0.59
	Room temperature***	0
	50°C	0.04*****
Incubation with enrichment reagent 2 on a Thermoshaker	No shaking***	0
	400 rpm	0.74
Time and speed of centrifugation	1 min, 8,000 g	1.31
	1 min, 10,000 g	1.57
	1 min, 16,000 g	1.37
	2 min, 5,000 g	1.79
	3 min 10,000 g	1.59
	3 min, 16,000 g	1.25
	10 min, 500 g	2.33
	15 min, 5,000 g	0.99
	15 min 16,000 g	0.29
	30 min, 3,000 g	1.25
	30 min 5,000 g	0.44
	30 min, 10,000 g	0.36
	30 min 16,000 g***	0
	60 min, 16,000 g	−0.13*****
Removal of residuals of supernatant	Washing with 1 ml RNase-free H2O	3.50
	No washing	0.53
	Spin down for 10 s***	0
Dissolving of the pellet for direct downstream applications	D1 (ddH2O)	n.d.
	D2 (PBS)	n.d.
	D3 (RIPA)	n.d.
	D4 (50 mM tri-natriumcitrat)	n.d.
	D5 (100 mM tri-natriumcitrat)	n.d.
	D6 (150 mM tri-natriumcitrat)	n.d.
	D7 (200 mM tri-natriumcitrat)	n.d.
	D8 (50 mM EDTA)***	n.d.
	D9 (100 mM EDTA)	n.d.
	D10 (500 mM EDTA)	n.d.
	D11 (1 M EDTA)	n.d.
	D12 (PBS + 50 mM EDTA)	n.d.

*For the development of an improved exosome enrichment method, a plasma volume of 500 µl was used. **CD values were calculated from the formula: C_q(x)-C_q(y), in which x is any variant tested and y is the best variant within the subgroup of the tested reagents and parameters (single boxes). The difference shows the magnitude of the aberrance of a variant from the best variant. The lower the CD value is the better is the quantity and quality of the PCR product. Hence, 0 means the best value. ***The selected best variations of reagents and parameters are in bold. Variations of results similar to the best variation that ****showed a minimal data improvement but were not confirmed by a further intensification of the parameter, or *****significantly increased the experiment duration/complexity, were not chosen for further testing. n.d., not determinable.