Use of Real-Time Polymerase Chain Reaction for the Detection of Fetal Aneuploidies - Clinical Applications of PCR - page 94

3. Methods
3.1. DNA Extraction of Amniotic Fluid Samples
1. Spin 1 mL of amniotic fluid at 16,000 g in a benchtop micro-centrifuge for 15 s to

pellet the amniotic cells.

2. Discard the supernatant and resuspend the cell pellet in 500 µ L of water.

3. Wash once by centrifuging the cells as in step 1 . Discard the supernatant.

4. Add 60 µ L of Chelex 100 resin to the pellet and resuspend the cells by vortexing.

5. Incubate the samples for 20 min at 57°C.

6. Incubate the samples in a dry heat block at 100°C for 8 min.

7. Prepare the sample for use in the real-time PCR analysis by heating to 95°C for

5 min. Following this, spin the samples at 16,000 g in a benchtop micro-centrifuge
for 2 min to pellet the resin. Carefully remove the aqueous DNA solution, taking
care not to disturb the resin pellet ( see Note 7 ).

8. Use 2 µ L of this DNA solution in a final reaction volume of 25 µ L for the PCR

analysis ( see Notes 8 and 9 ).

3.2. Performing Real-Time PCR
1. It is advisable to include at least one control sample of known karyotype in the

analytic run ( see Note 10 ).

2. Each sample should be analyzed at least in triplicate ( see Note 11 ).

3. The preparation of the real-time PCR reactions should be carried out on ice.

4. The real-time PCR amplification is carried out in a total volume of 25 µ L ( see

Note 12 ) containing 2 L of the sample DNA solution ( see Note 13 ), 300 n M of

each primer ( see Note 14 ), and 200 n M of each probe ( see Note 15 ) at 1X con-
centration of the Universal PCR reaction mix.

µ

5. This is prepared by first pipetting the PCR reaction mixture into the reaction

wells. Follow this step by adding 2 L of the sample DNA solution ( see Note 16 ).

Carefully seal the reaction plate with the optical adhesive cover. Centrifuge at
1000 g at 4°C for 1 min to spin down any droplets and remove air bubbles.

µ

6. Immediately start the real-time PCR cycler with the emulation mode off

( see Note 17 ).

7. Following an initial incubation at 50°C for 2 min to permit Amp Erase activity,

and another at 95°C for 10 min to allow activation of Ampli Taq Gold and
denaturation of the genomic DNA, use the following cycle conditions: 40 cycles
of 1 min at 60°C and 15 s at 95°C.

3.3. Analysis of Real-Time PCR Data
1. Perform the experimental analysis with a baseline setting of 3-22 cycles ( see

Note 18 ). Check the replicate curves for uniformity in the amplification plot view
( see Note 19 ) and for final fluorescence ( see Note 20 ).

2. Analyze four different thresholds at R n -values of: 0.2, 0.3, 0.45, and 0.675, as

indicated in Fig. 2 and Table 1 ( see Note 21 ). Export the C T result files and
examine the data using an Excel spreadsheet.