DETERMINING SPECIFIC THYROID TRANSCRIPTS
IN PERIPHERAL BLOOD:
A SINGLE CENTER STUDY EXPERIENCE
Makazlieva T, Eftimov A, Vaskova O, Tripunoski T,
Miladinova D, Risteski S, Jovanovic H, Jakovski Z
Tanja Makazlieva and Aleksandar Eftimov contributed equally to this study
and are considered first coauthors.
*Corresponding Author: Tanja Makazlieva, Ph.D., Institute of Pathophysiology and Nuclear Medicine,
Medical Faculty, Mother Teresa Street, No. 17, 1000, Skopje, Republic of Macedonia.
Mobile: +389-75-313-665. E-mail: tmakazlieva@medf.ukim.edu.mk or tmakazlieva@gmail.com
page: 13
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RESULTS
In our study, expression for the GAPDH gene was detected
in all 57 samples. From all 57 samples, seven TSHR
and/or Tg expression levels were undetectable, three were
from HC (in two cases both target genes were undetectable
in peripheral blood, and in one only Tg-mRNA was undetectable).
From the patients included in the examination,
one from the TCb group had undetectable Tg-mRNA, only
one from the TCs group had undetectable levels for both
target genes, and two other patients with advanced stages
of the disease had undetectable TSHR-mRNA. The highest
and lowest mean values of ΔCt = (CtTSHR-CtGAPDH) was in
TCr and TCs, respectively (12 ± 1.8 vs. 9.6 ± 2.6). The
statistical analysis revealed significant difference between
the mean values ΔCt = (CtTSHR-CtGAPDH) for all four groups
(χ2 = 13.312; df = 3, p = 0.004) (Figure 1 and Table 1).
Additional intergroup analysis of the mean ΔCt = (CtTSHR-
CtGAPDH) revealed significant difference between: a) TCs/
TCr (Z = –2.231; p = 0.026); b) TCs/HC (Z = –3.416; p =
0.0006); c) TCb/TCr (Z = –2.341; p = 0.019) and d) TCb/
HC (Z = –2.803; p = 0.005) (Table 2).
We have analyzed the statistical significance among
values of ΔCt = (CtTSHR-CtGAPDH) between two groups of patients with incomplete response to treatment (TCs+TCb)
and two groups composed of patients with excellent response
to treatment and healthy individuals (TCr+HC).
Analysis revealed significant differences between the two
combined groups (Z = –3.928; p = 0.00008) (Table 3 and
Figure 2).
Within the four research intergroups, comparison for
mean ΔCt = (CtTg-CtGAPDH) values was also performed. The
highest and lowest mean value for ΔCt = (CtTg-CtGAPDH)
was detected in the TCr group and TCs with 10 ± 2.6 vs.
6.17 ± 5.37, respectively (Table 4 and Figure 3).
Further intergroup analysis related to mean values of
ΔCt = (CtTg-CtGAPDH) revealed statistical significant differences
between: a) TCs/TCr (Z = –1.964; p = 0.049); b)
TCs /HC (Z = –2.453; p = 0.014) (Table 5). Within the
research comparison between two combined groups for
mean ΔCt = (CtTg-CtGAPDH) values was performed (Table
6 and Figure 4). Using the 2–ΔΔCt method, according to the previously
described quantification by Livak and Schmittgen [14], we
detected that TCs patients expressed TSHR-mRNA by a
8.57-fold higher level than HC individuals. The TCs patients
expressed TSHR by a 5.37-fold higher level than TCr patients,
TCb patients expressed TSHR by a 14.17-fold higher
level than HC individuals, and TCb patients expressed TSHR
by an 8.88-fold higher level than TCr patients (Table 7).
Whereas for Tg-mRNA, we found a 6.6-fold higher change
in expression in the TCs group compared to HC, an 8.34-fold
higher change in expression in the TCs group than TCr, a
1.53-fold change in expression in TCb and HC, and a 1.93-
fold change in expression in TCb compared to TCr (Table 7).
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