DNA extraction from single cells

As with every DNA based assay the quality of the DNA used in the assay has a massive effect on the quality of the resulting data. When dealing with DNA extracted from blood or tissue homogenates this does not pose a real problem. There is a large selection of commercially available kits and protocols which result in a high concentration of good quality DNA. DNA obtained through this route performs very well in real-time reactions.

However, DNA quality can be a major problem when investigating individual cells. Many commercially available kits do not cater for the low quantity of DNA and small volumes involved when working with individual cells. When we applied a standard single cell lysis protocol for real-time PCR amplification, considerable variability was observed in the absolute copy number measurement. Partial lysis of the cell and poor DNA extraction are the most likely explanation. If the study requires the relative quantification, then this does not pose such a huge problem, because inefficient extraction is unlikely to be template-specific. When measuring the absolute amount of mtDNA in a sample, partial lysis poses a real problem. If not all mtDNA molecules are available for amplification then it becomes difficult to draw meaningful conclusions.

To examine the true effect of lysis conditions on the measurement of absolute copy number, different lysis conditions should be considered. Cell lysis buffers can contain EDTA, as this is a magnesium chelating agent its effect on subsequent real-time reactions should be understood. Historically individual cell lysis reactions have been kept at as small a volume as possible to maximize the final DNA concentration. DNA purification methods are not 100% efficient. When measuring the quantity of mtDNA in a sample, a percentage loss can mean any subsequent conclusions are flawed. As such, post-lysis purification is not regularly performed. Table 11.4 shows

Table 11.4 Components and concentrations of different lysis buffers used to determine the effect of lysis efficiency on the detectable mitochondrial copy number.

Lysis buffer

Lysis buffer without EDTA

ABI GeneAmp® PCR buffer plus

50 mM Tris-HCl, pH8.5 1mM EDTA, pH8 0.5% Tween 20 100 ^g Proteinase K

50 mM Tris-HCl, pH8.5 0.5% Tween 20 100 ^g Proteinase K

ABI GeneAmp® PCR buffer (1X) 0.5% Tween 20 100 ^g Proteinase K

Table 11.5 mtDNA copy number measurements from skeletal muscle fibers dissected from 20 ^m cryostat sections and lyzed using different lysis methods. TOT shows the total mtDNA copy number measured using a region of ND1 and WT shows the wild-type mtDNA copy number measured using a region of ND4. The % Deln value shows the percentage deletion calculated using both measurements. Percentage deletion values should be zero as the fibers are from a control individual without an mtDNA deletion.

(A) Lysis buffer Copy number:

TOT

WT

% Deln

1530

32200

-2008.4

538

27800

-5070.9

757

1950

-157.1

19400

2050

89.4

6350

42800

-573.9

6210

12200

-1866.6

13900

1300

90.6

23200

5010

78.4

20000

957

95.2

8830

42100

-376.5

25500

8350

67.2

1790

33800

-1784.6

(B) Lysis buffer without EDTA

Copy number:

TOT

WT

% Deln

18000

19800

-9.9

51200

13400

73.9

37800

11500

69.4

19000

50400

-164.7

15800

12900

18.3

18600

10100

45.7

34600

13200

61.7

30000

12500

58.5

46800

13300

71.5

48600

14800

69.5

14500

45200

-212.1

12100

11100

8.2

(C) ABI GeneAmp® PCR buffer plus

Copy number:

TOT

WT

% Deln

766

761

0.36

1600

9430

-488.2

1120

2420

-116.0

2670

2990

-12.1

2550

3630

-42.3

9780

3200

67.3

1320

15800

-1095.5

3270

4210

-28.9

2440

11500

-373.6

1290

1530

-18.3

(D) Lysis buffer then purification

Copy number:

TOT

WT

% Deln

8190

8300

-1.4

33800

6030

82.1

59

2740

-4548.4

9890

7910

20.0

7270

7640

-5.1

29700

4230

85.7

10100

17300

-71.6

375

2740

-630.1

91100

16700

-83.3

11000

40500

-268.9

8460

7520

11.1

55900

64600

-15.6

different lysis buffers and Table 11.5 show the effect the use of each buffer has on the mtDNA copy number measured from skeletal muscle fibers taken from 20 pm cryostat sections. What can be clearly observed is that the inclusion of EDTA in the buffer increases the variability observed, whereas a post-lysis purification markedly decreases the copy number measurement.

Lysis temperature and incubation times are yet another variable that can have a large effect on the quantity and quality of DNA extract from a single cell. Using the lysis buffer without EDTA (see Table 11.4), lysis conditions over two temperatures and two incubation times have been investigated.

Table 11.6 Effect of lysis temperature and incubation time on mtDNA copy number measured from control skeletal muscle fibers taken from 20 ^m cryostat sections.

2 hour incubation at:

16 hour incubation at:

37°C

55°C

37°C

55°C

15200

18500

17800

23000

7640

19500

20700

20400

22100

16300

12400

18000

15400

15400

17100

37200

20700

16800

27200

29800

15400

24400

15900

19500

21400

19800

51200

22400

15300

15400

19800

24300

15600

14900

23200

25100

15600

22200

22500

27900

17800

16900

14900

26000

16600

18500

21600

31300

Incubation at 37°C and 55°C and for both 2 hour and 16 hour-time periods showed some variation in the copy number measured from single skeletal muscle fibers taken from 20 ym cryostat sections. Incubating at 55°C for 16 hours appears to generate a higher and more reproducible copy number value (Table 11.6).

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