Molecular Biology Reports (2019) 46:5057–5062

https://doi.org/10.1007/s11033-019-04959-5

ORIGINAL ARTICLE

Assessment of the expression level of miRNA molecules using

a semi‑quantitative RT‑PCR approach
Shima Andoorfar1 · Seyed Ali Hosseini Tafreshi1 · Zahra Rezvani1

Received: 10 March 2019 / Accepted: 27 June 2019 / Published online: 8 July 2019
© Springer Nature B.V. 2019

Abstract
Type 2 diabetes is one of the most prevalent diseases, which increases resistance to insulin in target tissues. The measure-
ment of miRNAs quantity is a molecular approach for diagnosis of diabetes. miRNAs are small non-coding RNA strings
of 21–23 long nucleotides that act as inhibitors in proteins translation. Several methods including Northern blot, qRT-PCR
and Microarray have been used for diagnosis of miRNA molecules. Real time PCR is an expensive and accurate quantitative
method that is widely used in miRNA studies. The miR-21 is an important miRNA in diabetes. In this study, for the first
time, a semi-quantitative protocol was developed to quantify different amounts of a synthetic miR-21. In addition to semi-
quantitative method, the miR-21 quantity was determined by quantitative method in several patients with type 2 diabetes and
healthy people. The results indicated that there was a direct relationship between the amount of synthetic miR-21 and the
intensity of the PCR bands. We also showed that the expression of miR-21 in people with type 2 diabetes increased compared
to healthy people. The results were observed by both quantitative and semi-quantitative methods. The real-time RT-PCR was
more sensitive than semi-quantitative PCR in identification of miRNAs. However, semi-quantitative PCR method benefited
from higher simplicity and lower costs for defining general patterns of miRNA expression.

Keywords Type 2 diabetes · miR-21 · Semi-quantitative PCR · Real-time RT-PCR

Abbreviations glucose production are contributed in prevalence of hyper-

miRNA MicroRNA glycemia. The prevalence of metabolic diseases especially
PTEN Phosphatase and tensin homolog obesity, dyslipidemia and type 2 diabetes are increasing all
PI3K Phosphoinositide 3-kinases over the world [1]. It seems that type 2 diabetes creates the
IL-6 Interleukin 6 biggest epidemic in human history. According to The World
TNF-α Tumor necrosis factor alpha Health Organization, nowadays 190 million people are dia-
IL-1B Interleukin 1 beta betic in the world and more than 350 million people suffer
PBS Phosphate-buffered saline from impaired glucose tolerance (IGT). Predictions shows
that 552 million people of the world especially in develop-
ing countries will get diabetes mellitus until 2030 [2]. Type
Introduction 2 diabetes will occur if the target tissues are resistant to
insulin or lose sensitivity to insulin. Various factors such
Diabetes is one of the most prevalent chronic diseases, which as genetic defect in insulin receptor, the presence of insulin
is resulted due to complicated reactions between genetic and antagonists and disruption of intracellular pathways tend to
environmental factors that increase blood glucose. Due to decrease the sensitivity of tissues to insulin. Due to resist-
diabetes mellitus etiology, decrease of insulin secretion, ance of tissues to insulin, the glucose uptake in muscle tis-
decrease of glucose consumption by cells and increase of sue and lipid are decreased and as a result, the glucose level
increases in blood. Nevertheless, the successful diagnosis
of disease in early stages could lead to effective treatment,
* Seyed Ali Hosseini Tafreshi
sahosseini@kashanu.ac.ir and delaying the disease progression with a high degree
of success. Nowadays, blood parameters and biomarkers
1
Biotechnology Division, Department of Cell and Molecular are used in screening people with a high risk for diabetes.
Biology, Faculty of Chemistry, University of Kashan, Markers can be enzyme, hormone, protein and nucleic acids
Kashan, Iran

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including RNA, DNA and miRNAs [3]. miRNAs are small
non-coding molecules with 21–23 nucleotides in length that
act as suppressors of gene expression. These regulators of
gene expression were first discovered by Ambros and his co-
workers during studying the role of gene lin-14 in C. elegans
[4]. Recent studies have shown that miRNA molecules could
regulate gene expression via controlling the mRNA stability
or disruption [5].
Today, miRNA molecules have been known as a main
regulator of gene expression and controller of biologic pro-
cesses, including cell proliferation, apoptosis, tissue differ-
entiation and pathologic processes. Moreover, changes in
biogenesis and the function of these molecules are found
to cause or contribute to the progress of special types of
disease in human [6]. The miRNA molecules compared to
other types of RNAs are more stable, and could be deter-
mined using a simple blood test [7]. This makes the miRNAs
as proper molecular biomarkers for diagnostic purposes.
Zamptaki et al. previously found a negative correlation
between some types of miRNAs and type2 diabetes [8].
Among the various miRNA molecules reported by differ-
ent authors, miR-21 in known as one of the most important
miRNA molecules in diabetes, which often had higher levels
and contributed in a wide range of diabetic and non-diabetic
diseases [9]. Dey et al. introduced miR-21 as the molecular
bridge between increased amount of glucose and expression
of pten gene [10]. It is also shown that pten gene negatively
regulates insulin through signaling pathway PI3K in lipid
cells 3T3-L1 [11].
Various diagnostic approaches have been used for detec-
tion of miRNA molecules, including northern blot, real-time
PCR, microarray and RNA-seq techniques. These methods,
in different degrees, mainly suffered from disadvantages
such as high cost, being time-consuming, producing poorly
resolution data and need for special infrastructures and spe-
cialized laboratory personnel or a large amounts of total
RNA or samples.
The real-time PCR is considered as a golden standard for
detection of various miRNAs in biological studies. Nowa-
days, real-time PCR is the method of choice for evaluating
changes in the expression of various miRNAs in biology and
medicine, especially for diagnostic purposes, and to contrib-
ute in disease treatment. Although this approach offers a few
advantages, it might not be readily available, when resources
are limited. The application of this method might be limited
mainly due to the high costs of the instruments and SYBR
Green-based master mixes used for real-time PCR. The com-
plexity of the method and the need for skilled technicians are
also among this method’s drawbacks.
The objective of this study was to evaluate the efficiency
of a semi-quantitative PCR protocol as a less expensive anal-
ysis of miRNAs. Semi-quantitative PCR was previously used
to determine the relative abundance of mRNA molecules,
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Molecular Biology Reports (2019) 46:5057–5062
and to our knowledge, it was not used to quantify miRNAs.
In this work, we developed a semi-quantitative PCR method
to quantify and evaluate the level of a synthetic miR-21 and
real miR-21 in healthy and diabetic RNA samples. The
results were compared with those of real-time PCR method.
Materials and methods
Sampling
Blood samples of 9 healthy and 12 diabetic people (age
range 40–70 years) were provided courtesy of Dr. Vakili
laboratory in Kashan, Iran. The participants consent was
obtain for research participation. The test was conducted on
peripheral blood mononuclear cell (PBMC) of fresh blood
samples. In order to isolate PBMC, Ficoll solution (Inno,
lymphodex Train) and PBS (Phosphate-buffered saline) was
used. Briefly, 2 ml PBS was mix with equivalent volume of
blood sample. One ml Ficoll was poured in a new Falcon
tube and then slowly-diluted blood with PBS added to the
Ficoll solution. The tubes were centrifuged at 4000 rpm for
20 min in 4 °C. The white rings of PBS were collected, and
added with the equal amount of PBS. Then centrifuged at
3000 rpm for 10 min in 4 °C.
RNA extraction and cDNA synthesis
Precipitated cells with a little amount of PBS were trans-
ferred to 1.5 ml microtube and 1 ml of TRizol (Yekta Equip
Co.) was added and vortexed. The tube kept in ambient tem-
perature for 5 min. Then, 200 µl chloroform was added and
left at ambient temperature for 10 min. The tube was centri-
fuged at 1300 rpm for 15 min at 4 °C. The upper phase con-
taining RNA was transferred to new tube, and 600 µl cold
isopropanol was added. The tube was inverted several times,
kept in ambient temperature for 10 min, and centrifuged at
1200 rpm for 13 min at 4 °C. After removing the superna-
tant, the white RNA pellet was washed with 1 ml of alcohol
75% and left air-dried. Then, the sample was solved in 50 µl
DEPC-treated water. The quality and quantity of extracted
RNA was checked by a spectrophotometer. The extract was
electrophoresed on 1% agarose gel.
DNA contamination was removed from all RNA sam-
ples by treating the samples with DNasel (Thermo Fisher
Scientific). Equal amounts of RNA of all healthy or patient
samples (500 ng) were used for cDNA synthesis. One µl
stem loop primer, 1 µl buffer (5×), 0.5 µl RiboLock RNase
inhibitor, 1 µl dNTP (10 mM) and 0.5 µl Revert Aid Reverse
Transcriptase (Thermo Fisher scientific) were mixed in a
microtube (0.2 ml). The synthesis was performed by a ther-
mal cycler (BioRad). The amplification steps included one
cycle of 5 min at 25 °C, 60 min at 42 °C and 5 min at 70 °C.
Molecular Biology Reports (2019) 46:5057–5062
In order to provide the calibration curve and evaluating the
efficiency of the semi-quantitative method, the synthetic
miR-21 (UAG​CUU​AUC​AGA​CUG​AUG​UUGA) was used
in various amounts (­ 103, ­106, ­109 and 1­ 012 copy number)
for cDNA synthesis.
The primer design and semi‑quantitative PCR
The miR-21-5p sequence was downloaded from a miRNA
database (www.mirba​se.org). Primers were designed by
online PrimerQuest Tool (https​://eu.idtdn​a.com). The qual-
ity of designed primers was analyzed by OligoAnalyzer Tool
software. The primer sequences used in this study were pre-
sented in Table 1.
Semi-quantitative PCR was done using different amounts
of cDNA of synthetic miR-21 or cDNA of RNA samples.
One µl forward (Fp21) and reverse (Rp21) primers were
used. The PCR operation was also conducted for measur-
ing U6 (as an internal control gene) in diabetic and healthy
samples using Fpu6 and Rpu6 primers. For each sample,
PCR was repeated three times. The reaction contained 1 µl
of each cDNA samples, 0.5 µl of each primers, 5 μl Taq
DNA Polymerase 2× Master Mix Red (Amplicon Co.) and
3 μl dd water in a final volume of 10 μl.
Before the main reactions, the PCR conditions includ-
ing thermal conditions and the number of cycles and the
cDNA concentrations were optimized. During the main PCR
cycles, temperature conditions including one initial dena-
turation cycle (3 min at 95 °C) followed by 35 cycles with a
denaturation steps for 5 s at 95 °C and a combined annealing
and extension step for 35 s at 61 °C. The PCR products were
electrophoresed on agarose 2.5%, stained with Ethidium bro-
mide and photographed. The analysis of band intensity was
done by ImageJ software (version 1.46r). The graphs were
drawn by GraphPad Prism software (version 6,). The rela-
tive expression of miR-21 in a real sample was defined as
the intensity mean of miR-21 bands divided by the intensity
mean of U6 bands in that sample. A t test was applied to test
whether there was difference between the means of diabetic
or healthy sample. One-way ANOVA was performed to ana-
lyze the means of band intensities resulted from different
Table 1  Primers used in this Primer Gene
study
Slp21 miR-21
Rtp u6 U6
Fp21 miR-21
Rp21 miR-21
Fpu6 U6
Rpu6 U6
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amounts of synthetic miR-21. The calibration curve were
drawn in Excel software to evaluate the efficiency of semi-
quantitative PCR.
Real‑time PCR
The results of semi-quantitative PCR was evaluated and
compared with a standard quantitative method. The expres-
sion of miR-21 in real samples determined using real-time
PCR. The real-time PCR was done using the same tube or
thermal conditions as in semi-quantitative PCR. The quanti-
tative RT PCR was performed by real-time PCR instrument
(Applied Biosystems StepOne™) and mi-real-time Eva-
Green Master Mix (Metabion). The relative expression of
miR-21 was calculated according to an equation previously
described by Pfaffl [12].
Results and discussions
Efficiency of semi‑quantitative PCR
The efficiency of semi-quantitative PCR in measuring the
relative expression of miRNAs was evaluated. Different con-
centrations of cDNA were prepared from a synthetic miR-
21 used as template in PCR. After a preliminary optimiza-
tion series of PCR the best annealing temperature (61° C)
and number of cycles (30 cycles) were selected for the next
series of PCR. As indicated from calibration curve in Fig. 1,
there was a good correlation between the mounts of syn-
thetic miR-21 and the intensity of PCR bands ­(R2 = 0.962).
Based on these results, the method had an acceptable sensi-
tivity to detect miRNA.
Semi‑quantitative PCR method to evaluate relative
expression of miR‑21 in real samples
Total RNA samples were evaluated spectrophotometrically
and on agarose gel before cDNA synthesis. As indicated in
Fig. 2, the presence of ribosomal RNA bands (18 s and 28 s)
along with the results of spectrophotometry (data not shown)
Sequence (5′ → 3′)
GAA​AGA​AGG​CGA​GGA​GCA​GAT​CGA​GGA​AGA​AGA​
CGG​AAG​AAT​GTG​CGT​CTC​GCC​TTC​TTT​CTC​AAC​
ATC​
CGC​TTC​ACG​AAT​TTG​CGT​GTCA​
CGC​CTA​GCT​TAT​CAG​ACT​GA
CGA​GGA​AGA​AGA​CGG​AAG​AAT​
GCT​TCG​GCA​GCA​CAT​ATA​CTA​AAA​T
CGC​TTC​ACG​AAT​TTG​CGT​GTCAT​
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Fig. 1  Calibration curve resulted from plotting the band intensity

against different copy numbers of synthetic miR-21 (­ 103, ­106, ­109 and
­1012 copy number). The diagram revealed a good correlation between
the number of miR-21 used in semi-quantitative PCR and the inten-
sity of resulting bands in electrophoresis

Fig. 3  Semi-quantitative analysis of miR-21 expression (a) and

expression profile (b) normalized by U6 gene as the internal control
in healthy or diabetic samples

Fig. 2  Gel electrophoresis of total RNA samples. Visualization of

total RNA isolated from diabetics blood (D) or healthy (H) samples
run on 1% agarose gel. g DNA is genomic DNA

Fig. 4  Relative expression of miR-21 determined by real-time PCR.

reveal the integrity of RNA samples. Accordingly, the samples
quality was appropriate for cDNA synthesis.
For evaluating the relative expression of miR-21, the inten-
sities of miR-21 bands in diabetic or healthy samples were
divided by the intensities of the internal control (U6 gene)
bands of corresponding sample. The results from Fig. 3 indi-
cated the relative expression of miR-21 in diabetic samples
increased by about two times compared to healthy samples.
Real‑time PCR method to evaluate relative
expression of miR‑21 in real samples
In order to confirm the results of semi-quantitative method,
quantitative method was used to determine of the gene
Diabetic samples showed a 71-fold increase in miR-21 expression
level compared to healthy controls
expression profile. In this research, miR-21 and internal con-
trol U6 were measured in healthy and type 2 diabetic people
by a quantitative method previously described by Pfaffl [12].
The efficiency of primers was validated by standard curves
and melting curves (data not shown). The results of real-time
PCR (Fig. 4) revealed that the relative expression of miR-21 in
diabetics was significantly higher with about 71-fold increase
compared than those in healthy samples.

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Discussion
The miRNA expression profile in blood circulation sys-
tem could contribute to development of biomarkers. The
miRNA biomarkers are important in diagnosis, prognosis
and even treatment of different diseases, such as cardio-
vascular, different types of cancers, diabetes, and arthritis.
Recent studies have shown that miRNAs played roles in
stimulation of insulin resistance in cells that that resulted
in diabetes. The first paper on the importance of miRNA
biomarkers in type 2 diabetes was published by Zampe-
taki et al. (2010) who showed that a decrease in miR-126
could be considered as a risk factor for diabetes. The study
was conducted on 800 people. The role and importance of
other miRNAs as risk factors in type 2 diabetes have been
investigated. The miR-21 is a multipurpose miRNA and
plays important roles in several physiologic and patho-
logic processes, such as inflammation, cancer, cardiovas-
cular disease and diabetes [13]. Moreover, the relationship
between miR-21 and type 2 diabetes in animal and experi-
mental models has been studied. It has been shown that
miR-21 decreased in liver of ob/ob rates which had symp-
toms of obesity and type 2 diabetes [14]. The expression of
miR-21 decreased in endothelial cells obtained from type 2
diabetic samples. The in vitro studies showed that miR-21
was low in fat cells treated with a high insulin or glucose
concentrations [15]. Thus, the change in expression of a
miRNA, such as miR-21 may be important for diagnosis
or treatment purposes in different diseases especially for
resistance to insulin and incidence of type 2 diabetes.
The real-time PCR method is the standard and com-
monly used procedure to study the expression of miRNAs
in biological studies. In this study, the role of miR-21 as
a potential biomarker, an alternative and more cost-effec-
tive method compared to the expensive methods, such as
real-time PCR is developed. The specificity and relative
sensitivity of semi-quantitative PCR was evaluated by a
synthetic miR-21, and then by miR-21 extracted from real
blood samples. This study was conducted on 12 people
with type 2 diabetes and 9 healthy people with ages ranged
from 37 to 80 years. The results showed that by both
semi-quantitative and real-time PCR methods, the rela-
tive expression of miR-21 in PBMC of diabetic samples
increased compared to healthy control. The difference in
miR-21 expression between diabetic and healthy samples
was statistically significant. It should be noted that quan-
titative method was more sensitive in measuring miR-21
so that, semi-quantitative method showed a 2-fold increase
in miR-21 concentration in diabetic samples. However, the
expression variation change was about 71 times higher
when the miR-21 quantified with real-time PCR method.
5061
Semi-quantitative analysis is considered a reliable method
for RNA expression study using qRT-PCR. The advantage
of this gel-based PCR method is offering a clear and visual
“band-based” answer that is applicable for analysis of de
novo expression of genes. However, one of the main limita-
tions of semi-quantitative approach as an end-point analysis
of RNA might be the need for running a preliminary series
of PCR to optimize the annealing temperature and number
of cycles to set-up the main series of PCR. The PCR condi-
tions should be fine-tuned to amplify proper concentrations
of PCR products. This could provide good band visuali-
zation on agarose gel without over-saturation. The semi-
quantitative PCR is less sensitive compared with real-time
method, especially when the expression variations are very
small. Therefore, some quantification information could be
lost. On the other hand, semi-quantitative methods would be
more cost efficient than quantitative PCR to analyze multiple
miRNAs with multiple housekeeping genes. This method is
easily applicable when there is limited access to real-time
PCR instruments.
Overall, the results showed that semi-quantitative PCR
could be used as a high-resolution method for finding gen-
eral patterns of miRNAs expression, and for diagnosis pur-
poses. The semi-quantitative approach is capable of quanti-
fying higher number of miRNAs.
Acknowledgements This work was financially supported by graduate
study of University of Kashan under Grant No. 572212/04.
Compliance with ethical standards
Conflict of interest The authors declare that they have no conflict of
interest.
Ethical approval This study was granted by the Aklaghezisti Ethical
Committee at University of Kashan. All procedures were in accordance
with the approved ethical standards of the Ethical Committee.
Informed consent Written informed consent obtained from all healthy
and diabetic people.
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