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200 µl L-Glutamin, 20 µl penicillin- streptomycin
and 200 µl phytohaemagglutinin. After incubation
at 37ºC for 72 hours, 200 µl Colchicine was added
to arrest the cells at metaphase. Following an
additional incubation at 37ºC for 30 minutes and
centrifugation at 20ºC for 8 min. at 1500 rpm the
supernatant was removed. The pellet was resuspended
with up to 10 ml hypotonic solution
(0.4% KCl solution) vortexed immediately. All the
samples were kept at 37ºC for 20 minutes and
again centrifuged at the same condition. After
removing supernatant from the samples, the pellet
which contains cells at metaphase, was
homogenised. Fixative solution (methanol and
acetic acid mixed with 3:1 ratio) was added and the
tubes were vortexed for the fixation of
chromosomes. Then samples were centrifuged after
adding up to 5 ml of fixative solution. Supernatant
was discarded from the samples and fresh fixative
solution was added to the tubes. This procedure
was repeated until the samples were clarified.
According to the cell density, up to 0.5 ml fixative
solution was added to the samples. Then samples
were homogenized and cells were lied onto slide
glasses, which were kept at 4ºC in distilled water
till they are used. After spreading the cells on the
slides, the samples were dried at room temperature
and kept overnight at 60ºC.
Y chromosome microdeletions in spontaneous abortions
Karyotyping
GTG (Giemsa-Trypsin) banding technique was
performed. When the banding of the chromosomes
was not successful, the protocol was repeated.
After staining, at least 20 metaphase plaques were
analysed for each sample (Figure 1).
Detection of Y chromosome microdeletions
DNA isolation from blood
DNA was extracted from 200 µl peripheral blood by
using High Pure PCR Template Preparation Kit
(Roche-Germany) according to the manufacturer’s
protocol.
Multiplex polymerase chain reaction
(multiplex PCR)
Table 1. Primers used for multiplex PCR and the length of amplicons.
MIX1 Amlicon
length (bp)
MIX2 Amlicon
length (bp)
For detection of Y chromosome microdeletions,
isolated DNA was amplified by multiplex PCR. AB
ANALITICA–The AZF Extension Kit, which is
recommended by European Andrology Association
was used in multiplex PCR. By using this kit, 13
different regions could be investigated at the same
time by performing 3 multiplex PCRs for each
sample. Three primer sets, each containing primers
that is unique to ZFX/Y locus which also exist in X
chromosome are shown in Table 1.
MIX3 Amlicon
length (bp)
ZFX/Y 495 ZFX/Y 495 DBY 689
SRY 472 SRY 472 ZFX/Y 495
sY 254 380 sY 95 303 SRY 472
sY 86 320 sY 117 262 sY 84 326
sY 127 274 sY 125 200 sY 134 301
sY 255 120 DFFRY 155
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