DNA extraction from paraffin embedded colorectal carcinoma samples: A comparison study of manual vs automated methods, using four commercially kits

Table 1 DNA isolation protocols

PureLink Genomic DNA mini kit (Invitrogen–ma-nual)	Time	iPrep genomic DNA kit (Invitrogen–aut-omated)	Time	High Pure FFPE DNA isolation kit (Rochemanual)	Time	MagnaPure LC DNA Isolation kit (Roche–automated)	Time
3 sections of 5 µm	5-10 min	3 sections of 5 µm	5-10 min	3 sections of 5 µm	5-10 min	3 sections of 5 µm	5-10 min
Deparaffinizatio-n using Xylol 3 times	3 x 10 min	Deparaffinization using Xylol 3 times	3 x 10 min	Deparaffinization using Xylol 3 times	3 x 10 min	Deparaffinization using Xylol 3 times	3 x 10 min
Lysis and digestion (20 µL of ProtK)	From 30 min to overnight	Sample lysis		Lysis and digestion (70 µL ProtK)	30-90 min	Sample lysis and binding
Binding DNA to silica membranes (columns)	15 min	Binding DNA to magnetic beads	30 min	Binding DNA to silica membranes (columns)	15 min	ProtK	30 min
Washing out contaminants	15 min	Magnetic separation of beads		Washing out contaminants	15 min	Binding DNA to beads
Eluting	3 min	Separation of liquid solutions from beads Washing beads eluting		Eluting	3 min	Washing out contaminants Washing beads eluting
~ 1.96 Euro/sample		~ 7.8 Euro/sample		~ 3.66 Euro/sample		~ 6.81 Euro/sample

Table 2 DNA concentration (ng/µL) using four commercially kits for DNA isolation

	PureLink Genomic DNA mini kit (Invitrogen–manual)	iPrep genomic DNA kit (Invitrogen–automated)	MagnaPure LC DNA Isolation kit (Roche–automated)	High Pure FFPE DNA isolation kit (Roche–manual)
Mean	157.24	8.72	20.39	86.64
Standard deviation	62.99	6.40	21.19	43.84
Minimum	37.60	0.70	0.30	4.20
Maximum	316	29.80	121	168.90

Table 3 DNA quality¹ using four commercially kits for DNA isolation

	PureLink Genomic DNA mini kit (Invitrogen–manual)	iPrep genomic DNA kit (Invitrogen–automated)	MagnaPure LC DNA Isolation kit (Roche–automated)	High Pure FFPE DNA isolation kit (Roche–manual)
Mean	1.80	1.78	1.84	2.19
Standard deviation	0.04	0.14	0.52	0.37
Minimum	1.67	1.42	0.99	1.60
Maximum	3.10	2.25	4.36	3.10

¹Quality–OD: A260/A280.

Table 4 Correlation between DNA concentration¹ and clinicopathological aspects of colorectal cancer

Characteristics		Number	DNA concentration PureLink Genomic DNA mini kit (manual)		P value
			≤ 150 ng/µL	> 150 ng/µL
Age	≤ 60	20	12	8	0.373
	> 60	26	11	15
Gender	Male	26	15	11	0.373
	Female	20	8	12
Macroscopic aspect	Polypoid	10	4	15	0.00103
	Ulcero-infiltrative	36	19	8
Microscopic aspect	G1	13	3	10	0.043
	G2 + G3	33	20	13
Localization	Proximal colon	8	5	3	0.892
	Distal colon	21	11	10
	Rectum	17	7	10
Depth of infiltration (pT stage)	T2-T3 T4	40 6	20 3	20 3	> 0.992
Lymph node metastasis (pN stage)	Present (pN1-3)	17	13	4	0.063
	Absent (pN0)	29	10	19
Lymph node ratio	< 0.15	36	17	19	0.723
	≥ 0.15	10	6	4

¹Manual PureLink Genomic DNA mini kit-Invitrogen.

²Chi square test.

³Fisher’s exact test. G1: Well differentiated; G2: Moderately differentiated; G3: Poorly differentiated.

Table 5 Correlation between DNA concentration¹ and clinicopathological aspects of colorectal cancer

Characteristics		Number	iPrep genomic DNA kit (magnetic beads)		P value
			≤ 10 ng/µL	> 10 ng/µL
Age	≤ 60	20	12	8	0.093
	> 60	26	22	4
Gender	Male	26	19	7	0.993
	Female	20	15	5
Macroscopic aspect	Polypoid	10	3	7	0.00043
	Ulcero-infiltrative	36	31	5
Microscopic aspect	G1	13	4	9	< 0.00013
	G2 + G3	33	30	3
Localization	Proximal colon	8	6	2	0.522
	Distal colon	21	17	4
	Rectum	17	11	6
Depth of infiltration (pT stage)	T2-T3 T4	40 6	28 6	12 0	0.112
Lymph node metastasis (pN stage)	Present (pN1-3)	17	14	3	0.313
	Absent (pN0)	29	20	9
Lymph node ratio	< 0.15	36	25	11	0.253
	≥ 0.15	10	9	1

¹Automated iPrep genomic DNA kit-Invitrogen.

²Chi square test.

³Fisher’s exact test. G1: Well differentiated; G2: Moderately differentiated; G3: Poorly differentiated.

Table 6 Correlation between DNA concentration¹ and clinicopathological aspects of colorectal cancer

Characteristics		Number	High Pure FFPE DNA isolation kit (manual)		P value
			< 100 ng/µL	≥ 100 ng/µL
Age	≤ 60	20	12	8	0.993
	> 60	26	15	11
Gender	Male	26	15	11	0.993
	Female	20	12	8
Macroscopic aspect	Polypoid	10	8	17	0.123
	Ulcero-infiltrative	36	19	2
Microscopic aspect	G1	13	3	10	0.0023
	G2 + G3	33	24	9
Localization	Proximal colon	8	5	3	0.962
	Distal colon	21	12	9
	Rectum	17	10	7
Depth of infiltration (pT stage)	T2-T3 T4	40 6	24 3	16 3	0.642
Lymph node metastasis (pN stage)	Present (pN1-3)	17	8	9	0.353
	Absent (pN0)	29	19	10
Lymph node ratio	< 0.15	36	23	13	0.273
	≥ 0.15	10	4	6

¹Manual High Pure FFPE DNA isolation kit-Roche.

²Chi square test.

³Fisher’s exact test. G1: Well differentiated; G2: Moderately differentiated; G3: Poorly differentiated.

Table 7 Correlation between DNA concentration¹ and clinicopathological aspects of colorectal cancer

Characteristics		P value	MagnaPure LC DNA Isolation kit (magnetic beads)		P value
			< 20 ng/µL	≥ 20 ng/µL
Age	≤ 60	20	12	8	0.383
	> 60	26	12	14
Gender	Male	26	12	14	0.383
	Female	20	12	8
Macroscopic aspect	Polypoid	10	8	2	0.043
	Ulcero-infiltrative	36	16	20
Microscopic aspect	G1	13	4	9	0.023
	G2 + G3	33	23	10
Localization	Proximal colon	8	3	5	0.602
	Distal colon	21	11	10
	Rectum	17	10	7
Depth of infiltration (pT stage)	T2-T3	40	20	20	0.442
	T4	6	4	2
Lymph node metastasis (pN stage)	Present (pN1-3)	17	5	12	0.033
	Absent (pN0)	29	19	10
Lymph node ratio	< 0.15	36	18	18	0.723
	≥ 0.15	10	6	4

¹Automated MagnaPure LC DNA Isolation kit-Roche.

²Chi square test.

³Fisher’s exact test. G1: Well differentiated; G2: Moderately differentiated; G3: Poorly differentiated.