Keywords
Dura Mater - Nucleic Acids - Gene Expression - RNA
Palavras-chave
Dura-máter - Ácidos Nucleicos - Expressão Gênica - RNA.
INTRODUCTION
The dura mater, the meninge that lines the central nervous system externally, is classified
as fibrous, due to a large number of collagen fibers, making it a thick and resistant
membrane.[1] The dura mater has two leaflets, an internal and an external leaflet, which have
an embryological origin from the neural crest cells and the mesoderm, respectively.[2] The main functions of the dura mater are isolation, mechanical protection and stabilization
of the central nervous system. In addition, the dura mater is richly vascularized
and innervated, mainly by the branches of the trigeminal nerve.[3] As the brain has no sensitive nerve endings, all intracranial sensitivity is located
in the dura and blood vessels, which appear to be involved in the pathophysiology
of several forms of headache.[4] In addition to being involved in headache attacks, the blood vessels that are related
to the dura mater are involved in different diseases and pathological manifestations.[3]
However, in order to understand the pathophysiological mechanisms of some diseases,
molecular analyzes are applied. To perform molecular techniques, it is necessary to
extract from the tissue molecules, such as nucleic acids and proteins, which are employed
in subsequent techniques. High-quality RNA is required for various techniques, including
sequencing, reverse transcription quantitative polymerase chain reaction (RT-qPCR),
microarray, etc.[5] Extraction and purification of RNA from fibrous tissues are challenging because
it is a very rigid tissue with a low amount of nucleic acids and proteins. In addition,
the number of cells in such tissue is generally very low, which leads to low RNA yield.[6]
There are many commercial products available; however, most of the time RNA extraction
kits do not bring specifications/adaptations to the different existing tissue types,
often leading to a low-yielding RNA without precision and reliability of results.
Not only in the case of fibrous tissues but some adaptations have already been made
in other types of tissue, such as adipose tissue, since many protocols routinely used
to isolate RNA from adipose tissue result in partially degraded RNA and/or low RNA
yield or the small RNAs are lost in the process.[5]
[7] However, for peculiar types of tissue, there is a lack of information to guide the
researcher in the preservation and purification protocols.
Therefore, the aim of the present article was to evaluate different combinations of
commercially available kits for dura mater maintenance and RNA purification in order
to establish a suitable combination between stock and purification reagent and evaluate
the viability of RNA through the repeatability of gene expression.
METHODS
Animals
All experiments were developed according to the guidelines of the Animal Ethics Commission
of the Universidade Federal de Pernambuco. Eight adult Wistar rats (between 8 and
10 weeks old) were purchased at the animal testing facility at the Academic Center
of Vitória at the Universidade Federal de Pernambuco. Before surgeries to remove the
dura mater, the animals were individually conditioned in polypropylene cages, stored
in rooms under standardized laboratory conditions of temperature (22 ± 2°C), relative
humidity (45 ± 5%), exhaust system with air renewal, luminance of 60 lux, and circadian
cycle of 12 hours in light period and 12 hours in dark period. During the period of
biological maintenance, the animals were fed with commercial feed and water ad libitum.
Withdrawal of dura mater
The animals were anesthetized with ketamine (115 mg/kg) and xylazine (10 mg/kg), followed
by tissue perfusion with 0.9% saline solution. To perform the dura mater collection
procedure, the calvaria was removed through an incision in the occipitofrontal direction,
the dura mater underlying the overlying bone disk was removed with the help of an
18-cm Molt detacher (Quinelato, Rio Claro, SP, Brazil). Two dura mater samples were
collected from each animal, weighing between 5.0 and 8.6 ηg. The samples removed were
quickly preserved in the following stabilization solutions: TRIzol (Thermo Fisher
Scientific, Waltham, MA, USA; cat. number 15596026) or RNAlater (Thermo Fisher Scientific, Waltham, MA, USA; cat. number AM7021). Then, samples were
frozen at - 80°C for subsequent RNA extraction and purification.
Dura mater RNA purification
RNA isolation was performed in duplicate using the following kits: TRIzol Reagent
(Thermo Fisher Scientific, Waltham, MA, USA; cat. number 15596026); RNeasy Mini Kit
(Qiagen, Hilden, Mettmann, Germany; cat. number 74104); Direct-zol (Zymo, Irvine,
CA, United States; cat number R2070T), and ReliaPrep (Promega, Madison, WI, United
States; cat. number: Z6110). The kits were tested for stored samples TRIzol and RNAlater, according to the compatibility of each of them with solvents. The samples were quantified
on the NanoDrop 2000 spectrophotometer (Thermo Scientific). The manufacturer protocol
was followed, with minor modifications. A pretreatment was adopted for all kits, in
which the samples were placed outside the freezer 24 hours before RNA extraction,
being maintained at room temperature (19°C to 21°C) to assure complete thawing of
all samples, and to allow the TRIzol reagent to act for sample digesting. Additionally,
samples preserved in RNAlater were incubated for 10 minutes at 60°C in a water bath before extraction to dilute
the crystals in the solution. In the first step of separation in the purification
protocol, all samples were submitted to a centrifugation time of 5 minutes at 12,000 × g, at 4°C.
cDNA preparation and real-time PCR evaluation
For cDNA synthesis, the QuantiTect Reverse Transcription Kit (Qiagen, Hilden, Mettmann,
Germany; cat number 205311) was used following its standard protocol, which is divided
into two main steps: the removal of genomic DNA (gDNA) and the synthesis of cDNA.
In the first step, 0.5 µl of the gDNA Wipeout Buffer was added to 3.0 µl of the total
RNA. The first cycle was performed on the Thermocycler (Applied Biosystems, Waltham,
Massachusetts, United States, 96-well thermal cycler, 0.2 ml, ref. 4375786), at a
temperature of 42°C for 2 minutes, then the tubes were immediately placed on ice.
For cDNA synthesis, 1.5 µl of a mix containing 0.25 µl of reverse transcriptase, 1 µl
RT Buffer, and 0.25 RT µl primer mix was added to the sample. The samples went through
the second cycle of 15 minutes at 42°C, 3 minutes at 95°C and 4°C ∞. Quantification
was performed on the NanoDrop 2000 spectrophotometer (Thermo Scientific, Wilmington,
DE, United States).
The qPCR reaction was performed by the StepOne Real-Time PCR-System (Applied Biosystems,
Foster City, CA, USA) with the GoTaq qPCR Master Mix kit (Promega). Each PCR was performed
in duplicates, in 96-well plates. They were used with fixed volumes, 5 μL SYBR (asymmetrical
cyanine dye – SYBR Green I) and 1 μL of Carboxy-X-Rhodamine (CXR). A total of 0.2
μL of the beta-actin reference gene (Actb), Rn_Actb_1_SG assay (QuantiTect Primer
Assay, Qiagen, Germany) was added. To complete the volume of 9 μL of reagents, 2.8
μL of water was added. Finally, 1μL of the cDNA sample was added, totaling the final
reaction volume with 10 μL. For cycling, the fast protocol was used with 1 initial
cycle of 95°C for 2 minutes for activation of Taq DNA polymerase, followed by 40 cycles
of denaturation (95°C for 3 seconds) and annealing (60°C for 30 seconds). To assess
the quality of the cDNA over the days, three qPCR plates were performed for 3 weeks.
During the 3 weeks in which the plates were being performed, the cDNA samples were
kept at −80°C.
Data processing
The Ct values were expressed as mean and standard deviation (SD), calculated based
on the values obtained over the 3 weeks. In the analysis of intrarepeatability, the
mean and SD between duplicates were calculated over the 3 weeks.
RESULTS
Dura mater RNA purification
The present study aimed to optimize and evaluate yield and purity levels of dura mater
total RNA isolation protocols based on commercially established methods. An additional
step of thawing the sample 24 hours before the procedure facilitated the RNA purification
once the phase separation after the centrifugation at the first steps of the extractions
became more evident. [Table 1] shows the quantitative measurements of the total isolated RNA, the mean, and SD
of the Ct values over the 3 weeks. In all samples, the elution volume was the same,
regardless the tissue mass.
Table 1
Quantitative measurements of the RNA purification, and RT-qPCR evaluation on average
Ct over 3 weeks
|
Storage reagent
|
Purification kit
|
Sample
|
Tissue (ηg)
|
RNA yield (ηg/μL)
|
260/280
|
260/230
|
β-actin (ΔCt)
|
Intrarepeatability mean (ΔCt)
|
|
TRIzol
|
TRIzol
|
R1A
|
8.4
|
13.0
|
1.55
|
2.45
|
37.2 ± 15.2
|
36.7 ± 0.5*
|
|
R1B
|
7.5
|
36.5
|
1.42
|
1.54
|
36.2 ± 18.7
|
|
TRIzol
|
Direct-zol
|
R2A
|
7.6
|
4.3
|
1.57
|
- 0.04
|
26.2 ± 3.1
|
26.3 ± 0.1
|
|
R2B
|
6.9
|
9.4
|
1.73
|
- 0.08
|
26.4 ± 3.1
|
|
TRIzol
|
ReliaPrep
|
R3A
|
8.6
|
16.1
|
1.84
|
1.83
|
28.3 ± 3.2
|
28.8 ± 0.5
|
|
R3B
|
6.3
|
7.2
|
1.80
|
2.00
|
29.4 ± 3.3
|
|
TRIzol
|
RNeasy
|
R4A
|
6.3
|
5.5
|
1.69
|
0.35
|
30.1 ± 3.2
|
28.5 ± 1.6
|
|
R4B
|
8.1
|
9.5
|
1.83
|
0.58
|
26.9 ± 2.8
|
|
RNAlater
|
ReliaPrep
|
R5A
|
5.9
|
5.0
|
1.89
|
- 6.36
|
26.2 ± 2.8
|
25.6 ± 0.6
|
|
R5B
|
7.1
|
8.5
|
2.15
|
5.52
|
25.0 ± 2.9
|
|
RNAlater
|
RNeasy
|
R6A
|
5.0
|
19.2
|
2.00
|
1.21
|
27.2 ± 2.9
|
27.8 ± 0.6
|
|
R6B
|
6.2
|
21.3
|
1.80
|
1.02
|
28.4 ± 3.2
|
Notes: All samples were maintained at −80°C until the gene expression analysis.
*Samples showed expression only in the first of the three tests performed with β-actin.
The RNA yield could not be related to the sample weight, since high RNA yield could
be obtained even in samples with lower mass input, varying from 5.5 to 35.5 ηg/μL.
Regarding the RNA purity against DNA contamination, 50% of samples fit in the acceptable
range (Abs 260/280 = 1.80 to 2.00). Only 1 sample was above the limit, and the others
reaching values as low as 1.42. However, the deviation was significant for protein
contamination, with most samples showing very low or even negative records in some
samples. Only one sample fit in the range (Abs 260/230 = 2.0–2.2). The TRIzol/TRIzol
combination was tested twice (in duplicate), but only the second experiment was included
in the table, as only these two showed Ct values.
To understand the impact of these numbers in the gene expression, samples were submitted
to cDNA synthesis and evaluated in RT-qPCR test for β-actin expression.
β-actin gene expression in the dura mater
The dissociation curve at the end of the RT-qPCR showed the peak temperatures of the
curve in the range of 86.03°C to 86.22°C ([Figure 1]). In the 1st week of RT-qPCR, the best Ct average among duplicates, with a value of 22.4, was
obtained using the RNAlater/ReliaPrep method, followed by RNAlater/RNeasy, with an average of 24.4. The TRIzol/ReliaPrep and TRIzol/RNeasy methods showed
a small range of variation, with an average of 24.2 and 25.1, respectively. Regarding
the 3-week evaluation, TRIzol/ReliaPrep showed the best values compared wth the RNAlater/ReliaPrep method. The RNAlater/RNeasy method, on the other hand, showed better results compared with the TRIzol/RNeasy
method ([Table 1]).
Figure 1 Dissociation curve at the end of the RT-qPCR.
Figure 2 Intra-repeatability of the samples over three weeks.
The gene expression evaluation was performed three times, with an interval of one
week between each evaluation. The TRIzol/TRIzol method showed no reproducibility in
RT-qPCR. Despite the highest yield, this combination could not be associated with
good gene expression results, since β-actin was undetectable in the 2nd and 3rd weeks of testing ([Figure 2]). It is interesting to note that the methods based on TRIzol/Direct-zol and RNAlater/ReliaPrep showed negative purity values, which suggest protein contamination, but
the levels of gene expression were the best in both. In fact, the RNAlater/ReliaPrep method showed the best Ct average over the 3 weeks ([Figure 2]).
Lower Ct values were observed in the 1st week for all samples, with a significant increment in the 2nd and 3rd weeks ([Figure 2]). An intrarepeatability analysis demonstrated that the kits had similar Ct values
between the duplicates, varying from 25.0 to 30.1 along the 3 weeks. In this interval,
there was an increase of 4 cycles in Ct values compared with the 2nd week, and 1 cycle for the 3rd week of evaluation ([Figure 2]). The mean and SD of the Ct values are shown in [Table 1]. TRIzol/TRIzol has no evaluation since there were no Ct values throughout all analyzes.
Amplification was successful in the case of RNAlater-based methods. However, for the TRIzol method, the use of a commercial kit based
on affinity membrane method is suitable to assure higher yield and reproducibility
in gene expression analysis.
DISCUSSION
Traditionally, the levels of RNA gene expression in some tissues are low, making subsequent
techniques difficult.[8] Although commercial kits include some specifications for different types of tissues
in their protocols, the dura mater was not included in any of them, considering that
it is not a tissue usually studied. The adaptations proposed for the dura mater RNA
extraction protocols brought the addition of an incubation step before the beginning
of the extraction, which implied the advancement of the cell lysis process. The RNA
evaluation over 3 weeks allowed us to assess the quality and preservation of the material,
in addition to the efficiency of each kit. These results were shown to be better in
membrane-based protocols compared with purification with liquid-liquid kits.
There are several commercially available approaches for enriching RNA in biological
samples, but most protocols result in very low yields due to a lack of specificity
for the nature of the various existing tissues. Therefore, some protocol adaptations
were found in the literature, but none for the dura mater. The dura mater has a strong
relationship with neurovascular structures and, consequently, is an important tissue
to evaluate the molecules that are released in the CNS, potentially involved in the
pain mechanism. In addition, these optimizations do not include the evaluation of
the methods of RNA stabilizing.[6]
[7]
[9]
[10] In order to determine the quality of a given sample, a spectrophotometer meets the
demands with significant savings in time and money, short measurement cycle, and is
easy to use,[11] although other methods can be used with higher precision, but also at a higher cost.[6]
[7]
[10]
In the present study, the highest concentration values were obtained by the TRIzol
method, but the RNA purity was poor. This occurrence was already described for adipose
tissue processing, suggesting the chemical contamination of residues (proteins, traces
of organic solvents and salts) as responsible for the overestimated concentration
of nucleic acids. It also influences downstream analysis, leading to an increase in
Ct and SD values (5,7), as observed in the present study for the dura mater. Thus,
despite the low cost of the TRIzol method, it is important to note some inconveniences
regarding harmful odors, laborious handling, in addition to the unsatisfactory purity
that the method can present.
In contrast, the preservation in RNAlater provides nucleic acids of high quality and conserved morphology for diverse tissues,
besides not needing a quick-freezing system after immersing the sample. The method
based on RNAlater stabilization has a higher cost but does not emit toxic odors, as in the case of
TRIzol, in addition to presenting yields as good as those of TRIzol, with a low rate
of RNA contamination.[12] In addition, taking into account the data of purity, concentration, and Ct values,
the samples stored in RNAlater had the best results.
It is interesting to note that the samples with low levels of purity related to contamination
by proteins (260/230), like TRIzol/Direct-zol and RNAlater/ReliaPrep, showed the best Ct values for β-actin expression, which indicates that
the evaluation of RNA yield and concentration could have been misled in the spectrophotometer
evaluation.
The suggested adaptations may contribute to RNA extraction in human dura mater, since
there are no structural differences between tissues. Furthermore, the study of gene
expression of molecules present in the dura mater may contribute to elucidate mechanisms
of diseases that are related to this structure, such as migraine. In addition, the
physiopathogenic understanding of the behavior of structures and molecular units related
to the dura mater aims to contribute to the development of tools and answers that
clarify points in clinical research, and that these can contribute to application
in possible therapeutic targets.
LIMITATIONS OF THE STUDY
Although the adaptations suggested in the protocols are inexpensive, molecular biology
techniques are generally very sensitive, requiring well-equipped laboratories and
high-cost materials. Most of the data reported here are derived from dura mater samples;
analyzing other similar tissues can expand the effectiveness of the method. Overcoming
the challenges and limitations of RNA extraction protocols from specific tissues,
such as the dura mater, will increase the use of molecular biology techniques and,
thus, may contribute to the quality of analysis, providing better diagnostic tests.
