In . J. Mol. Sci. 2013, 14, 3456-3466; doi:10.3390/ijms14023456
In e na ional Jou nal o
Molecula Sciences
ISSN 1422-0067
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A icle
Valida ion o Re e ence Genes o he De e mina ion o Pla ele
T ansc ip Le el in Heal hy Indi iduals and in Pa ien s wi h he
His o y o Myoca dial In a c ion
Ka alin S. Zsó i 1, László Muszbek 1,2, Zol án Csiki 3 and Ami H. Shemi ani 1,2,*
1 Clinical Resea ch Cen e , Uni e si y o Deb ecen, Medical and Heal h Science Cen e ,
Deb ecen 4032, Hunga y; E-Mails: zska [email protected] (K.S.Z.); [email protected] (L.M.)
2 Th ombosis and Haemos asis Resea ch G oup o he Hunga ian Academy o Sciences,
Uni e si y o Deb ecen, Medical and Heal h Science Cen e , Deb ecen 4032, Hunga y
3 Depa men o In e nal Medicine, Uni e si y o Deb ecen, Medical and Heal h Science Cen e ,
Deb ecen 4032, Hunga y; E-Mail: [email p o ec ed]
* Au ho o whom co espondence should be add essed; E-Mail: [email p o ec ed];
Tel.: +36-30-6856608; Fax: +36-52-340011.
Recei ed: 31 Decembe 2012; in e ised o m: 21 Janua y 2013 / Accep ed: 30 Janua y 2013 /
Published: 6 Feb ua y 2013
Abs ac : RT-qPCR is he s anda d me hod o s udying changes in ela i e ansc ip le el
in di e en expe imen al and clinical condi ions and in di e en issues. No alida ed
e e ence genes ha e been epo ed o he no maliza ion o ansc ip le el in pla ele s.
The e y low le el o pla ele RNA and he elimina ion o leukocy e con amina ion
ep esen ed special me hodological di icul ies. Ou aims we e o apply a simple echnique
o sepa a e pla ele s o ansc ip le el s udies, and selec he mos s able e e ence genes
o pla ele s om heal hy indi iduals and om pa ien s wi h he his o y o myoca dial
in a c ion. We de eloped a simple, s aigh o wa d me hod o pla ele sepa a ion o
RNA isola ion. Pla ele ac i a ion was inhibi ed by using acid-ci a e-dex ose o
an icoagula ion and by p os aglandin E1. Leukocy e con amina ion was elimina ed by h ee
consecu i e cen i uga ions. Samples p epa ed by his me hod we e ee o leukocy es,
showed no inhibi ion in PCR eac ion and no RNA deg ada ion. The assay demands low
blood olume, which complies wi h he equi emen s o e e yday labo a o y ou ine.
Se en een po en ial e e ence genes we e in es iga ed, bu eigh o hem we e excluded
du ing op imiza ion. The s abili y o he emaining genes, EEF2, EAR, ACTB, GAPDH,
ANAPC5, OAZ1, HDGF, GNAS, and CFL1, we e de e mined by ou di e en desc ip i e
OPEN ACCESS
In . J. Mol. Sci. 2013, 14 3457
s a is ics. GAPDH, GNAS, and ACTB we e shown o be he mos s able genes in pla ele s
o heal hy indi iduals, while HDGF, GNAS, and ACTB we e he mos s able in pla ele s o
pa ien s wi h he his o y o myoca dial in a c ion. The esul s con i m ha da a
no maliza ion needs assessmen o app op ia e e e ence genes o a pa icula sample se .
Keywo ds: ansc ip le el; no maliza ion; pla ele ; e e ence gene; RT-qPCR
1. In oduc ion
Re e se ansc ip ion quan i a i e eal- ime PCR (RT-qPCR) is, a p esen , he mos sensi i e
me hod o he de ec ion and quan i a ion o low abundance mRNAs [1]. I is c ucial o con ol o
a ia ion be ween samples when measu ing mRNA exp ession. One app oach is o no malize o o al
RNA. This app oach needs a eliable RNA quan i ica ion me hod and ails o ake in o accoun he
a iabili y in e e se ansc ip ion and o he s eps o he measu emen [2]. The use o e e ence genes
(RGs) as an in e nal con ol is he mos common app oach o da a no maliza ion [3]. On he o he
hand, he use o a single in e nal RG o no maliza ion could lead o ela i ely la ge e o s [4]. RGs
used o he quan i ica ion o mRNA exp ession could a y wi h issue ype as well as wi h
physiological, pa hological and expe imen al condi ions, and hei alida ion and op imiza ion o
accu a e and ep oducible quan i a ion is essen ial [5]. P e ious epo s on pla ele mRNA exp ession
used con en ional RGs [6,7], bu de ails conce ning hei selec ion, alida ion, and in es iga ion o
s able exp ession we e no p o ided.
Ci cula ing pla ele s con ain an excep ionally small amoun o megaka yocy e de i ed mRNA. Low
mRNA concen a ion and con amina ion wi h leukocy es a e wo main hind ances o pla ele ansc ip
le el s udies. To dec ease he olume o whole blood equi ed o single indi idual ansc ip le el
s udies and o ampli y he s a ing pla ele mRNA, di e en ampli ica ion s a egies ha e been used [8].
Leukocy e con amina ion was dec eased by il a ion and magne ic leukocy e-deple ion [8–10]. These
echniques could no dec ease he olume o whole blood equi ed o he measu emen s o less han
40 mL. Toyama e al. succeeded in dec easing he equi ed olume o 20 mL, bu hey did no p o ide
i m e idence on e icien emo al o leukocy es om pla ele suspension (see Supplemen a y
Table S1) [11]. The aims o ou s udy we e: (1) o ind a simple p ocedu e o he sepa a ion o
pla ele s om o he blood cells, (2) o de elop a me hod ha equi es only low olume o blood o
mRNA quan i ica ion, and (3) o selec and alida e he mos s ably exp essed mRNAs om a panel o
se en een RGs which can be used in RT-qPCR expe imen s o he no maliza ion o ansc ip le el in
pla ele s o heal hy indi iduals and pa ien s wi h he his o y o myoca dial in a c ion. The exp ession
s abili y o he RGs was es ed by ou di e en app oaches desc ibed in he li e a u e [4,12–14] using
Re Finde algo i hm [15]. To ensu e expe imen al anspa ency, accu acy, and epea abili y, we
ollowed he MIQE guidelines [16].
In . J. Mol. Sci. 2013, 14 3458
2. Resul s
2.1. E alua ion o RNA In eg i y, Con amina ion and Inhibi ion
Ampli ica ion o each o he candida e genes was con i med by he appea ance o a single peak in
he RT-qPCR mel ing cu e analyses. P io o ca ying ou RT-qPCR eac ions, he in eg i y o all
RNA samples was examined using eal- ime PCR o e alua e he exp ession o he GAPDH gene [5].
RNA in eg i y was in he accep able ange o all samples. When SPUD amplicons we e added o each
qPCR eac ion in equal amoun , he eac ions demons a ed comple e absence o qPCR inhibi ion.
Whi e blood cell con amina ion o all samples was uled ou by nega i e esul s (Cq > 40) o eal- ime
PCR o g anulocy e-speci ic mRNA (CD15) and lymphocy e-speci ic mRNA (HLA-DQβ).
2.2. Exp ession Le el o Pu a i e Re e ence Genes
B2M, TBP, UBC, HMBS, PTMA, WIPI2, NCOA, and VAMP we e elimina ed om u he analyses
du ing op imiza ion. Exclusion c i e ia we e unaccep able e iciency, low exp ession le el, and he
p esence o non-speci ic p oduc s o p ime dime . Non-speci ic ampli ica ion and p ime dime can
alsely inc ease ansc ip le el, especially when in e cala ing dyes a e used o assess eal- ime PCR.
Figu e 1. Comp ehensi e gene s abili y in heal hy indi iduals (A), and in pa ien s wi h he
his o y o myoca dial in a c ion (B).
In . J. Mol. Sci. 2013, 14 3459
Figu e 1 shows he s abili y o e e ence genes o pla ele ansc ip le el s udy in heal hy
indi iduals and in pa ien s wi h he his o y o myoca dial in a c ion. S abili y is exp essed as he
geome ic means o esul s om ou di e en ypes o calcula ion. Candida e e e ence genes showed
Cq alues be ween 19 and 34. The ampli ica ion e iciency o analyzed genes was as ollows: CFL1
(102%), ACTB (108%), EEF2 (89%), HDGF (81.6%), GAPDH (117.3%), ANAPC5 (81%), GNAS
(83%), EAR (104%), OAZ1 (96%) (Supplemen a y Table S2). Acco ding o Vandesompele e al.,
minimum h ee in e nal con ol genes should be used o he co ec no maliza ion o RT-qPCR da a.
We ound GAPDH, GNAS, and ACTB o be he bes combina ion o e e ence genes o pla ele
ansc ip le el s udies in heal hy indi iduals, while HDGF, GNAS, and ACTB a e he bes o s udies
in pa ien s wi h he his o y o myoca dial in a c ion.
Figu e 2. Cyclo-oxygenase 1 (COX-1) ansc ip le el in pla ele s o pa ien s wi h he
his o y o myoca dial in a c ion. T ansc ip le els we e no malized o OAZ1 (A), o o
HDGF, GNAS, and ACTB (B).
In . J. Mol. Sci. 2013, 14 3460
To demons a e he use ulness o alida ed candida e e e ence genes in RT-qPCR, he exp ession
le el o cyclo-oxygenase 1 (COX-1) gene ansc ip in pla ele s o pa ien s wi h he his o y o
myoca dial in a c ion, ela i e o i s exp ession in a con ol, was in es iga ed, using he leas s able
gene (OAZ1) and he h ee mos s able e e ence genes o no maliza ion (Figu e 2A,B). COX-1 is
cons i u i ely exp essed in many issues and is esponsible o physiological p os anoid p oduc ion.
The mean ela i e le el o COX-1 mRNA in pla ele s no malized o OAZ1 was 10- old highe han
ha no malized o HDGF, GNAS, and ACTB.
3. Discussion
A ew me hods ha e been published o he p epa a ion o pla ele s ee o con amina ing
leukocy es o s udy mRNA exp ession. These me hods usually need high blood olume, and
ime-consuming p epa a ion s eps. In some cases, he absence o leukocy es om he inal pla ele
suspension was no , o was no adequa ely e i ied. Ou me hod o pla ele isola ion consis s o h ee
cen i uga ions (45 min) and needs only 12 mL o pe iphe al blood. Such pla ele p epa a ions we e
ee o g anulocy es and lymphocy es, as i was demons a ed by PCR-based me hods.
The RT-qPCR is accep ed as he me hod o choice o he accu a e and sensi i e quan i ica ion o
ansc ip le els. The de e mina ion o eliable e e ence genes is an essen ial s ep o analyzing
ansc ip le el using RT-qPCR. The e o e, i is necessa y o alida e he exp ession s abili y o he
con ol genes o speci ic o gans, pa ien g oups and/o expe imen al condi ions p io o i s use o
no maliza ion. I is a common p ac ice o use adi ional e e ence genes as in e nal con ols wi hou
e i ying hei alidi y. Based on p e ious s udies we in es iga ed se en een po en ially e e ence
genes and on he basis o op imizing s udies, nine genes we e selec ed o u he s udies. The
s abili ies o EEF2, EAR, ACTB, GAPDH, ANAPC5, OAZ1, HDGF, GNAS, and CFL1 genes we e
de e mined by ou di e en desc ip i e s a is ics.
Many analy ical p og ams ha e been de eloped o co ec sample- o-sample a ia ion in s udies o
he iden i ica ion o RGs and he ela i e quan i ica ion o ansc ip le el in a s udy popula ion. The
use o mul iple e e ence genes is he mos accu a e me hod. Bes Keepe , No mFinde and geNo m
p og ams a e usually p e e ed, because hey a e suppo ed by use - iendly so wa e. The majo
weakness o geNo m and he app oach de eloped by Sil e e al. [13] a e hei sensi i i y o
co- egula ion. Pai wise compa ison appa en ly ends o selec hose genes wi h he highes deg ee o
simila i y in hei exp ession p o ile. No mFinde app oach does no accoun o sys ema ic e o s
du ing sample p epa a ion. The Bes Keepe p og am conside s all genes showing a a ia ion in hei
amoun o s a ing ma e ial by he ac o wo o mo e as uns able, which migh limi i s use [17].
I has been epo ed ha h ee so wa e p og ams (geNo m, No mFinde and Bes Keepe ) esul ed
in di e en anking o de in an a emp o selec he mos s able e e ence genes [17,18]; we also
ob ained di e en esul s using ou di e en app oaches (Supplemen a y Figu es S1 and S2). I is a
common p ac ice o apply mo e han one s a is ical me hod o he selec ion o he mos s able
e e ence genes [18–20]. We employed he Re Finde p og am, which uses he mean o ou s a is ical
app oaches and anked he e e ence genes as a unc ion o hei means. Re e ence genes wi h he
lowe means we e conside ed mo e s able han he o he s.

In . J. Mol. Sci. 2013, 14 3461
We acknowledge ce ain limi a ions o ou s udy. Al hough a numbe o measu es we e in oduced
o keep pla ele ac i a ion and RNA deg ada ion a a minimum le el, he maximum ou -hou
s o age/ anspo a ion ime migh be a sou ce o a iabili y in ce ain condi ions. When a s udy is
ca ied ou on samples o pa ien s, i is di icul o minimalize he ime be ween blood d awing and
sample p ocessing. In he case o di e en disease condi ions he e ec o s o age/ anspo a ion on he
in eg i y o pla ele RNA should be e alua ed. Ano he limi a ion o he s udy could be he age
di e ence be ween he g oup o heal hy indi iduals and pa ien s wi h co ona y a e y disease.
Al hough, in con as o some o he issues, in whole blood no age dependence o gene exp ession was
obse ed [21], age di e ence migh in oduce a ce ain le el o unce ain y.
4. Expe imen al Sec ion
4.1. Subjec s
Twen y-one pa ien s (age 51–77, 69% male) wi h he his o y o myoca dial in a c ion we e
ec ui ed o he s udy. The diagnosis o myoca dial in a c ion was es ablished a he ime o i s onse
acco ding o he c i e ia o he Ame ican College o Ca diology and he Eu opean Socie y o
Ca diology. Pa ien s we e assessed o co ona y scle osis by co ona y angiog aphy. All pa ien s
demons a ed ≥50% s enosis a leas in one majo co ona y a e y o in one o hei b anches. Pa ien s
we e on p e en i e low dose aspi in he apy. Pla ele s we e also collec ed om se en appa en ly
heal hy indi iduals (age 26–54, 60% male) ha did no ake any medica ion.
4.2. Sample Collec ion and P epa a ion
Twel e mL blood samples we e collec ed in ubes con aining acid-ci a e-dex ose (Bec on
Dickinson, Schwecha , Aus ia) The ubes we e kep a ambien empe a u e and anspo ed wi hin
ou hou s om he clinical wa d o he analy ical labo a o y whe e i was immedia ely p ocessed. On
eaching he labo a o y p os aglandin E1 (100 nmol/L) was added o each ube, and samples we e
cen i uged wice a 150g, o 15 min, a 37 °C. The o o was allowed o decele a e wi hou using he
b ake. Each ime he uppe 2/3 o he supe na an was used o he nex s ep. A e a hi d
cen i uga ion (2500 pm, 15 min, 37 °C) he supe na an s we e comple ely emo ed. I is ad isable o
in e he ubes on a s e ile issue o one minu e and hen wipe ou hei walls om supe na an ’s
emnan s. Cen i uga ion is an ine i able s ep o pla ele isola ion, bu i could ac i a e pla ele s.
Special ca e was aken o p e en pla ele ac i a ion, which migh ha e inc eased RNA deg ada ion.
Due o i s low pH, an icoagula ion by acid-ci a e-dex ose, decele a ion o cen i uges wi hou b ake,
main enance o empe a u e a 37 °C du ing pla ele p epa a ion, and he addi ion PGE1, an e ec i e
inhibi o o pla ele ac i a ion all con ibu ed o keeping pla ele ac i a ion a a minimum le el. I is o
be no ed ha in p elimina y expe imen s we did no obse e any signi ican RNA deg ada ion caused
by up- o ou hou s s o age/ anspo a ion and pla ele p epa a ion.
RNA was isola ed om he pla ele pelle by QIAamp RNA Blood Mini Ki (Qiagen, Hilden,
Ge many) acco ding o he manu ac u e ’s ins uc ions. As measu ed by NanoD op 2000
spec opho ome e (The mo Fishe Scien i ic, Wal ham, MA, USA) he RNA concen a ions we e in
he ange o 2–4 ng/μL. Howe e , as NanoD op does no measu e RNA concen a ion below 5 ng/μL
In . J. Mol. Sci. 2013, 14 3462
p ecisely, we did no assign RNA concen a ion o he samples and in he case o all samples he same
olume o RNA p epa a ion was used o cDNA syn hesis. The in eg i y o all RNA samples was
examined by de e mining he GAPDH 3':5' signal a io [5]. The signal a io was a ound one ( ange:
0.96–1.02) demons a ing he high ex en o in eg i y. SPUD assay was used o check o inhibi ion in
each qPCR [22]. PCR analysis o each RNA sample was conduc ed o ensu e he absence o RNA
con amina ion om whi e blood cells [23]. All pa icipan s p o ided in o med consen ; he s udy was
app o ed by he E hical Commi ee o he Medical and Heal h Science Cen e , Uni e si y o Deb ecen.
4.3. Op imizing P ime Concen a ions and PCR E iciency
Six een non- ibosomal candida e e e ence genes we e selec ed om lis s o genes ecommended as
e e ence genes in wo big s udies [3,24]. In addi ion he Alu epea s we e also e alua ed as candida e
e e ence gene [25]. P ime s’ leng hs we e 19–25 nucleo ides (TIB MOLBIOL, Be lin, Ge many),
wi h a heo e ical Tm o 59–61 °C. The size o PCR amplicons anged om 64–150 base pai s. P ime s
we e designed o yield p oduc s spanning exon-exon bounda ies o p e en possible ampli ica ion om
con amina ing genomic DNA. PCR p oduc s we e subsequen ly esol ed in 2% aga ose gel o check
o speci ic size o he amplicon. Fo gel elec opho esis, samples we e esol ed a 80 V in a 2%
aga ose gel wi h 0.5× T is-bo a e/EDTA bu e s and s ained wi h e hidium b omide o isualize
p oduc s. The indi idual e iciency o each p ime pai we e ob ained by using s anda d cu e [26].
As indica ed by Pes ana e al. and he manu ac u e ’s manual, he e iciency be ween 50% and 120%
was accep ed [27,28].
Ou op imiza ion goals we e o iden i y he lowes p ime concen a ion ha s ill yields he lowes
quan i ica ion cycle (Cq), esul s in maximum luo escence and gene a es a single amplicon o co ec
size wi h p edic ed mel ing empe a u e. In hese expe imen s, all combina ions o six concen a ions
(100, 200, 300, 400, 600, 900 nM) o o wa d and e e se p ime s o se en een genes we e
used o gene a e op imal ampli ica ion plo s (Supplemen a y Table S2) [29]. These se en een
genes we e: GNAS (guanine nucleo ide-binding p o ein, alpha-s imula ing), ACTB (ac in, be a),
HDGF (hepa oma-de i ed g ow h ac o ), PTMA (p o hymosin, alpha), B2M (be a-2-mic oglobulin),
GAPDH (glyce aldehyde-3-phospha e dehyd ogenase), HMBS (hyd oxyme hyl-bilane syn hase),
TBP (TATA box binding p o ein), UBC (Ubiqui in C), EAR (exp essed Alu epea s), OAZ1 (o ni hine
deca boxylase an izyme 1), WIPI2 (WD epea domain, phosphoinosi ide in e ac ing 2), NCOA4 (nuclea
ecep o coac i a o 4), EEF2 (euka yo ic ansla ion elonga ion ac o 2), VAMP ( esicle-associa ed
memb ane p o ein), ANAPC5 (anaphase p omo ing complex subuni 5), and CFL1 (co ilin 1).
P ime and p obe cha ac e is ics a e shown in Supplemen a y Table S2. PCR e iciency (E),
coe icien s o de e mina ion (R2), and slope alues we e de e mined (Supplemen a y Table S2) using
i e se ial 2- old dilu ion poin s; Cq(s) we e plo ed e sus he loga i hm o dilu ion [26]. Minus RT
con ols, ha we e included o each un, we e uni o mly nega i e. PCR speci ici y o each gene was
de e mined by dissocia ion cu e analysis and gel elec opho esis. All p ime se s p oduced a
symme ical amplicon peak in mel ing poin analyses. In none o he samples was p ime -dime peak
and no- empla e con ol (NTC) eac ion (a nega i e con ol wi hou cDNA empla e) was obse ed.
The absence o p ime -dime was also e i ied by gel elec opho esis.
In . J. Mol. Sci. 2013, 14 3463
4.4. Re e se T ansc ip ion
RT was ca ied ou unde RNase- ee condi ions. Limi ed quan i ies o RNA dic a ed us o use ixed
olume (5 μL) o inpu RNA o each cDNA syn hesis [2]. RNA was e e se- ansc ibed o cDNA in
20 μL olume in he Ligh Cycle 480 (Roche) by 1s S and cDNA Syn hesis Ki (Roche). Fo he
subsequen RT eac ion, 0.8 μg (0.04 A260 uni s) oligio-p [dT]15 p ime , 1.6 μg (0.08 A260 uni s) andom
p ime p[dN]6, 20 uni s AMV e e se ansc ip ase (Roche) and 50 uni s RNase inhibi o we e added
and incuba ed a 25 °C o 10 min and hen a 42 °C o 60 min. We pe o med qPCR on RT iplica es
ins ead o qPCR echnical eplica es [30].
4.5. qPCR
Real- ime PCR using Ligh Cycle 480 SYBR G een I Mas e (Roche) was pe o med in he
Ligh Cycle . PCR eac ion consis ed o 10 μL Mas e Mix (2× concen a ion), di e en concen a ion
o p ime s as es ablished by op imiza ion (Supplemen a y Table S2), and 5 μL 10- old dilu ed e e se
ansc ibed o al RNA (in a inal olume o 20 μL). The ollowing ampli ica ion p og am was used:
hea ing o 10 min a 95 °C, 40 cycles o dena u a ion o 10 s a 95 °C, ollowed by 60 °C o 30 s
(55 °C in case o GAPDH) and 72 °C o one second. Subsequen ly, a dissocia ion cu e (mel ing
cu e) analysis was applied wi h one cycle a 95 °C o 15 s, 60 °C o 1 min and 0.5 °C amp a e o
95 °C o con i m speci ic ampli ica ion. Cq- alues we e co ec ed o PCR e iciencies wi h he
equa ion, Cq100% = CqLogE, (Cq100% = Cq a 100% e iciency). This way, he di e ences in
e iciency be ween di e en e e ence genes we e compensa ed.
4.6. Da a Analysis
Du ing he assessmen o a se o RGs, he implemen ed e alua ion me hod can be a sou ce o bias
ela ed o he assump ions unde lying each app oach. In an e o o minimize bias, we es ed he
exp ession s abili y o nine selec ed in e nal con ol candida e genes by ou di e en app oaches
ound in he li e a u e [4,12–14] by using Re Finde p og am [15].
ΔCq app oach in oduced by Sil e e al. compa es he ela i e exp ession o all pai wise
combina ion o genes wi hin each sample [13]. This compa ison p o ides in o ma ion on which pai s
show leas a iabili y and hence which gene(s) has he mos s able exp ession. The Bes Keepe
so wa e calcula es e e ence gene’s s anda d de ia ion (SD) based on aw Cq alues ega dless o
sample’s e iciency [14]. No mFinde analysis enables es ima ion o he o e all a ia ion o he
candida e no maliza ion genes [12]. The combined measu e o in a- and in e g oup- a ia ion is gi en
as a s abili y alue, which is an es ima ion o he a ia ion in he exp ession o candida e RGs. The
basic assump ion is ha a s able RG should ha e minimal a ia ion ac oss expe imen al g oups and
subg oups. Finally, geNo m is a Visual Basic Applica ion o Mic oso Excel, which uses an
algo i hm o calcula e M- alue, a ansc ip le el s abili y measu e, de ined as he mean pai wise
a ia ion o a gi en gene compa ed o he emaining es ed genes. S epwise exclusion o he e e ence
gene wi h he leas s able exp ession inally assigns he wo mos s able genes [4]. Re Finde is a
web-based ool o selec he mos s ably exp essed mRNAs among a panel o RGs. I in eg a es
geNo m, No m inde , Bes Keepe , and he compa a i e ΔC me hod o ank he candida e RGs. I
In . J. Mol. Sci. 2013, 14 3464
calcula es he RG anking based on each p og am, hen e alua es inal anking by assigning a sui able
weigh o an indi idual gene and calcula es he geome ic mean o hei weigh s.
5. Conclusions
Acco ding o ou knowledge, his is he i s s udy on he exp ession s abili y o candida e e e ence
genes in pla ele s. This s udy iden i ied g oups o genes sui able o accu a e no maliza ion o
RT-qPCR da a in he sample o heal hy indi iduals o pa ien s wi h he his o y o myoca dial
in a c ion. Ou esul s indica e ha GAPDH, GNAS, and ACTB a e he mos s able genes exp essed in
pla ele s o heal hy indi iduals and HDGF, GNAS, and ACTB we e iden i ied as he mos s able
e e ence genes exp essed in pla ele s om pa ien s wi h he his o y o myoca dial in a c ion. The
esul s on he exp ession o COX-1 mRNA clea ly demons a ed ha he selec ion o inapp op ia e RG
could lead o alse assessmen o he le el o a speci ic ansc ip . The selec ion o app op ia e RGs is
essen ial o pla ele ansc ip le el s udies.
Acknowledgemen s
Suppo om he Na ional O ice o Resea ch and Technology (Jedlik Ányos g an ,
NKFP-07-A1-2008-0127), om he Na ional Resea ch Fund (OTKA g an K 78386), om he
Hunga ian Academy o Sciences (MTA11003, TKI 227) and om he TÁMOP-4.2.2/B-10/1-2010-0024
p ojec is acknowledged.
Con lic o In e es
The au ho s decla e no con lic o in e es .
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