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TRANSCRIPT
Validation of appropriate reference genes for the normalization
of real-time quantitative RT-PCR data obtained from platelets of
post-myocardial infarction patientsAmir H Shemirani, Katalin S Zsóri, László Muszbek
Clinical Research Center, Debrecen University, Hungary. Thrombosis Hemostasis and Vascular Biology group of the Hungarian Academy of Sciencess
On reaching the laboratory PGE1 (100 nmol/L) was immediately added to each tube
and samples were centrifuged twice at 900 rpm (150g), 15 min, at 37 °C, without
breaking.
Each time the upper 2/3 of supernatant was used for next step. After a third
centrifugation (2500 rpm, 15 min, 37 °C) the supernatants were completely removed.
RNA was then isolated from the platelet pellet by QIAamp RNA Blood Mini Kit
(QIAGEN) according to manufacturer’s instruction. The very low level of RNA did
not alow us to measure its concentration in the samples. RNA integrity was
determined by GAPDH 3’:5’ signal ratio detection. The SPUD assay was used to
check for inhibition in each qPCR reaction.
Two-step RT real-time PCR
Five ng total RNA was reverse-transcribed to cDNA in 20 l volume in the
LightCycler 480 (Roche) thermal cycler by 1st Strand cDNA Synthesis Kit (Roche).
For the subsquent RT reaction, 0.8 g (0.04 A260 units) oligio-p [dT]15 primer, 1.6 g
(0.08 A260 units) random primer p[dN]6, 20 units AMV reverse transcriptase and 50
units RNase inhibitor were added and incubated at 25°C for 10 min and then at
42°C for 60 min. We implemented 3 RT replicates instead of qPCR replicates.
Real-time PCR using LightCycler 480 SYBR Green I Master (Roche) in 20 l on the
LightCycler was then performed. PCR reaction consisted of 10 l Master Mix (2x
concentration), different concentration of primers according to optimized
concentration (from 300 nM to 900 nM), and 5 ng reverse transcribed total RNA.
The following amplification program was used: heating for 10 minutes at 95°C, 40
cycles of denaturation for 10 seconds at 95°C, followed by 60 °C for 30 seconds and
72 °C for one second. Subsequently, a dissociation curve (melting curve) analysis
was applied with one cycle at 95 ºC for 15 s, 60 ºC for 1 min and 0.5 °C ramp rate to
95 °C to confirm specific amplification.
Figure 2. Average expression stability (M) of nine
candidate reference genes by geNorm analyses in
patients
Conclusions
We recommend ACTB and HDGF as stable RGs most suitable for gene expression studies of human platelets
after myocardial infarction. We propose the use of GNAS, OAZ1 and GAPDH average as RGs for the accurate
normalisation of qRT-PCR performed on normal platelets.
Our results clearly demostrated that the expression levels of RGs change in certain conditions. Thus, the selection of
RGs is important for platelet gene expression studies.
The use of these genes as RGs may further enhance the robustness of qRT-PCR in this model system.
Introduction
Reference genes (RGs) used for the quantification of mRNA expression could vary
with the experimental and disease conditions and their validation is a crucial
requirement. Previous reports on platelet mRNA expression used conventional RGs,
but no studies have been reported on checking the validity of these RGs. In other
words, the expression of RGs has not been thoroughly investigated in platelet. The aim
of our study was the establishment of RGs in platelets from healthy persons and in
patients with the history of myocardial infarction.
To ensure experimental transparency, accuracy, and repeatability, we followed the
MIQE guidelines (minimal information for publication of real-time qPCR
experiments). Materials and methods
12 mL blood samples were collected in ACD tubes from 21 patients who suffered
myocardial infarction. We also included seven healthy individuals into the study as
control. The tubes were stored at ambient temperature, and were transported within 4
hrs (also at ambient temperature) from the clinical ward to the analytical laboratory.
Primers’ lengths were 19-25 nucleotides, with a theoretical Tm of 59-61 ºC (exept
GAPDH with a Tm of 55ºC ). The amplicons’ sizes ranged from 75-250 bp. GenEX
software (MultiD Analyses AB, Göteborg, Sweden) was used to analyze the qPCR
data. Extensive optimization was performed for primers and probe optimization. All
primer pairs were chosen to span an exon-intron boundary to exclude amplification of
genomic DNA.
WBC contamination
Contaminating leukocytes were completely removed by three consequent
centrifugation as judged by PCR analysis.
Total RNA extraction
Figure 3. Ranking of candidate genes according to
NormFinder for patients
Figure 4. Average expression stability (M) of nine
candidate reference genes by geNorm analyses in
controls
Figure 5. Ranking of candidate genes according to
NormFinder for controls
Assessment of leukocyte depleted-platelet (LDP) RNA
purity
To monitor the effectiveness of leukocyte depletion,
we used a sensitive RT-PCR assay. RNA from platelet
preparations tested positive for a platelet-specific
mRNA (vWF) and negative for granulocyte-specific
mRNA (CD15) and lymphocyte-specific mRNA (HLA-
DQ ). Ethidium bromide-stained agarose gel contain-
ing Reverse transcription polymerase chain reaction-
amplified products of platelet mRNA was positive for
vWF and negative for CD15 and HLA-DQ after 40
cycles of polymerase chain reaction (Figure 1).
1) PLT vWF, 2) PLT CD15, 3) PLT HLA-DQ
4) NTC, 5) NRCWBC vWF 451-bp
WBC HLA-DQ 704-bp
Tm: 89
WBC CD15 370-bp
WBC HLA-DQb 704-bp
WBC CD15 370-bp
1 2 3 4 5MW
50bp
Figure 1. Reverse transcription polymerase chain
reaction-amplified products of platelet mRNA