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