Modified Spin Column-Based RNA Extraction Methods of Staphylococcus aureus using PureLinkA RNA Mini Kit and Basic Laboratory Instrument Muhammad Taufiq Hidayat1.
Endry Nugroho Prasetyo1 Department of Biology.
Faculty of Science and Data Analytics.
Institut Teknologi Sepuluh Nopember.
East Java.
Indonesia Correspondence:
Muhammad Taufiq Hidayat.
Laboratory of Microbiology and Biotechnology.
Department of Biology.
Institut Teknologi Sepuluh Nopember.
Sukolilo.
Surabaya Zip Code: 60111 Email:
muhammadtaufiqhidayat7@gmail.
Received: December 22, 2020 Revised: May 31, 2021 Accepted: Juni 1, 2021 Published: October 30, 2021 Abstract RNA extraction is an important process before gene expression assessment at the transcriptomic level.
RNA is a sensitive material to environmental factors such as temperature and contaminants, so the RNA extraction process generally requires sophisticated and expensive laboratory instruments.
In this study, we extract RNA from Staphylococcus aureus bacteria using the PureLinkA RNA Mini Kit.
The instruments used in this study are basic instruments such as a hand homogenizer and non-thermal The results of RNA extraction were visualized using agarose gel electrophoresis.
These results indicate that bacterial RNA extraction can be performed using the PureLinkA RNA Mini Kit even with inexpensive basic laboratory instruments.
Keywords Basic Laboratory Instrument.
PureLinkA RNA Mini Kit.
RNA Extraction.
Staphylococcus aureus.
DOI: 10.
33086/ijmlst.
This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
A2021 by author.
Muhammad Taufiq Hidayat & Endry Nugroho Prasetyo
INTRODUCTION
centrifuge again, forcing the wash buffer Total RNA extraction from bacterial through the membrane to removes any cells is used to analyze gene expression at the remaining contaminant from the membrane, transcriptional level.
High-quality total RNA leaving only the nucleic acid bound to the is a prerequisite for a variety of downstream silica gel.
Lastly, the column is put in a applications .
RNA quality and quantity centrifuge again, forcing the elution buffer are important factors for ensuring the through the membrane.
The elution buffer accuracy of gene expression analysis and removes the nucleic acid from the membrane other RNA-based downstream applications and the nucleic acid is collected from the .
The purity and integrity of RNA can bottom of the column.
impact the accuracy of techniques such as Staphylococcus aureus is an important Real-Time quantitative Polymerase Chain nosocomial Gram-positive pathogen that Reaction .
Spin Column-Based is one of causes various infectious diseases in humans, the RNA extraction methods that comprise of ranging from harmless localized skin lesions four stages namely lysis of cells, binding of to systemic infections, such as endocarditis, nucleic acid to silica gel membrane, washing the nucleic acid bound to the silica gel diseases .
Gram-positive bacteria have a membrane, and elution of the nucleic acid thick peptidoglycan layer and no outer lipid .
RNA extraction is an life-threatening First, the cell membrane was broken by a important step to assess genetic expression, lysis solution in order to free nucleic acid for example, to determine the effect of the from cell.
The binding solution that contains buffer solution and ethanol is added to a spin expression of S.
Generally.
RNA column, and the column is put in a centrifuge.
extraction uses commercial kits that permit Samples are lysed and homogenized in the for fast purification of high-quality nucleic presence of guanidinium isothiocyanate, a The success of commercial kits largely chaotropic salt capable of protecting the RNA relies on the spin columns assembled with from endogenous RNases .
The centrifuge solid-phase nucleic acid binding material forces the binding solution through a silica which allows easy binding, washing, and gel membrane inside the spin column.
The elution of nucleic acids in the purification nucleic acid will bind to the silica gel process .
membrane as the solution passes through if The extraction method should be subject the pH and salt concentration of the binding to various considerations such as budget, solution is optimal.
The column is put in a organism, and in particular, the aim of the Ina.
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: 73Ae80 Muhammad Taufiq Hidayat & Endry Nugroho Prasetyo experiment .
RNA extraction process Lysis and Homogenization Cell pellet was transferred to RNase-free expensive laboratory With 6 mL Lysis Buffer prepared with 2- limited laboratory instruments, we evaluate mercaptoethanol was added to sample using RNA extraction using PureLinkA RNA Mini RNase-free pipette tips.
Sample was vortexed Kit and basic instruments such as a hand at high speed until the cell pellet is homogenizer and non-thermal centrifuge.
completely dispersed and the cells appear
Sample was homogenized with hand
homogenizer (Mini Hand Homogenizer MT-
MATERIALS AND METHODS
Materials PureLinkA RNA Mini Kit (Thermo Fisher 13K) for 2 minutes.
Binding.
Washing, and Elution Scientific.
United State.
omprises of Lysis One volume 70% ethanol was added to Buffer.
Wash Buffer I.
Wash Buffer II, each volume of cell homogenate.
Sample was RNase-Free Cartridges, vortexed to mix thoroughly and to disperse Collection Tube.
, 2-Mercaptoethanol.
any visible precipitate that may form after aureus Culture, and Luria Bertani Broth.
The adding ethanol.
Sample up to 700 AAL instrument used are hand homogenizer (Mini .
ncluding any remaining precipitat.
was Hand Homogenizer MT-13K) and non- transferred to the Spin Cartridge .
ith the thermal centrifuge (BR Technologie.
Collection Tub.
Sample was centrifuged at aureus Cultivation and Cell Harvesting 8,000 rpm for 15 seconds Water.
Spin The flow-through was discarded harvesting was done based on .
A one- and the Spin Cartridge was reinserted into the loop single colony of aureus from same Collection Tube.
Steps .
ransfer to spin Mannitol Salt Agar and Luria Bertani Agar cartridge and centrifug.
was repeated until was transferred to Luria Bertani Broth.
the entire sample is processed.
Seven Cultured was incubated for 24 hours at 37oC.
hundred AAL Wash Buffer I was added to the Culture in Luria Bertani Broth was harvested Spin Cartridge.
Sample was centrifuged at by centrifuging at 4,000 rpm for 5 minutes 8,000 rpm for 15 seconds and removes supernatant.
The flow-through and the RNA Extraction Collection Tube was discarded.
Bacterial The Spin Cartridge was placed into a new manufacturerAos guide protocol (PureLinkA Collection Tube.
Five hundred L Wash RNA Mini Ki.
with modification.
Buffer IIwith ethanol was added to the Spin Cartridge.
Sample was centrifuged again at Ina.
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: 73Ae80 RNA Extraction was done based on the Muhammad Taufiq Hidayat & Endry Nugroho Prasetyo 8,000 rpm for 15 seconds The flow-through was discarded RESULTS The visualization of agarose gel under and the Spin Cartridge was reinserted into the same Collection Tube.
Steps Wash buffer II indicated by the formation of the single bands was repeated once.
The Spin Cartridge was pointed by arrow (Figure .
RNA
centrifuged at 8,000 rpm for 1-2 minutes to dry the membrane with attached the RNA.
The Collection Tube was discarded and the Spin Cartridge was inserted into a Recovery Tube.
50 L RNaseAeFree Water was added to the center of the Spin Cartridge.
Sample was incubated at room temperature for 1 minute.
The Spin Cartridge was centrifuged for 2 minutes at 8,000 rpm at room temperature to elute the RNA from the membrane into the Recovery tube.
Agarose Gel Electrophoresis The integrity of total RNA isolated and the extent of genomic DNA contamination electrophoresis .
The integrity and size of Figure 1.
Visualization of Agarose Gel under UV after 3 days stored at -40oC.
visualization is done in duplicate .
hown in wells 1 and .
The bands formed are shown in a and b.
RNA can be checked by denaturing agarose gel electrophoresis and ethidium bromide Unlike DNA.
RNA molecules This result indicated that PureLinkA RNA Mini Kit with basic laboratory instruments such as a hand homogenizer and structures .
5 L sample was mixed with non-thermal centrifuge can be used to extract 5 L nuclease-free water and 2 L loading total RNA from S.
aureus culture.
The result of RNA isolation was dye, then transferred to 1.
2% agarose gel Electrophoresis was run at 100V for 1 Agarose gel was dipped in Ethidium Bromide for 30 minutes and then visualized High Performance Ultraviolet Transilluminator (Thermo Fisher Scientific.
United State.
stored at -40oC for 3 days then was visualized Transilluminator determine whether the extracted RNA still survived or degraded after the storage period.
The results of the second electrophoresis are shown in Figure 2.
Ina.
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: 73Ae80 Muhammad Taufiq Hidayat & Endry Nugroho Prasetyo then processed through a spin cartridge containing a clear silica-based membrane to which RNA binds.
The binding mechanism of nucleic acid adsorption on the silica hydrogen bond, salt bridge and electrostatic force formed between the nucleic acid and the silica surface, and the solubility of nucleic acid .
Any impurities are effectively removed by subsequent washing.
The Figure 2.
Visualization of Agarose Gel under UV after 3 days stored at -40oC.
visualization is done in duplicate .
hown in wells 1 and .
The bands formed are shown in a and b.
purified RNA is then eluted in RNase-free water .
Yang et al.
also reported that the RNA isolated using e silica spin columnbased was relatively less contaminated by DISCUSSION The The success of RNA isolation is application of high concentration GuSCN influenced by several factors, including the .
/v 50%) in subsequent binding and choice of method and the availability of washing buffers also enhanced removing Rodriguez et al.
stated that denatured proteins and other inhibitors.
the extraction method should be subject to RNA various considerations such as budget, organism, and the aim of the experiment.
PureLinkA RNA Mini Kit is designed for fast laboratory instruments, we evaluate RNA purification of high-quality nucleic acids extraction using PureLinkA RNA Mini Kit based on the Spin Column-Based method.
and basic instruments such as a hand The PureLinkE RNA Mini Kit provides a homogenizer and non-thermal centrifuge.
simple method for isolating high-quality the manufacturer's protocol .
, it is stated RNA.
that homogenization can be done with 3 homogenized in the presence of guanidine alternative tools, namely the Homogenizer, isothiocyanate, and ethanol is added to the Syringe and needle, and Rotor-stator.
The sample .
Guanidine isothiocyanate is a Homogenizer provides highly consistent chaotropic salt capable of protecting the RNA results and is more convenient than other from endogenous Rnases .
The sample is homogenization methods .
Xu et al.
Bacterial With Ina.
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The results of this study increased from 35.
4Ae37.
6 (Ct value.
without indicate that the use of a non-thermal homogenization to 37.
8Ae40.
2 (Ct value.
centrifuge can be an effective alternative for with homogenization.
The lysis step is RNA extraction if a thermal centrifuge is not combined with a homogenization step that available in the laboratory.
includes denaturation with a guanidine- Novelty thiocyanate containing buffer to inactivate This study reports a new RNA isolation the RNases released from the cell .
strategy using simple laboratory instruments.
Wever et al.
indicate that the important According to the PureLinkA RNA Mini Kit factor that determines the results of RNA guide, homogenization can use one of 3 extraction is the buffer volume added during alternatives, namely 1.
Homogenizer.
Syringe and needle.
Rotor-stator.
Another recommendation of the homogenization important process in RNA isolation is The results of this study prove that In the manufacturerAos guide, it hand homogenizer can be used for the sample is recommended to use a temperature- homogenization process in the cell lysis controlled centrifuge because RNA is sensitive to high temperatures.
However, in Another factor that affects the RNA instruments are not available, so in this study, centrifuge is a vital tool in the RNA alternative instruments that are widely extraction process.
The centrifuge forces the available in public laboratories are used, binding solution through a silica gel namely non-thermal centrifuge and hand membrane inside the spin column, forces The results of this study wash buffer through the membrane to indicate that this simple instrument can be removes any remaining contaminant from the used to isolate S.
aureus RNA.
Even after membrane, and forces elution buffer through being stored for 3 days at -40oC, the results of the membrane .
In this study, we used a electrophoresis still showed that the isolated non-thermal RNA RNA was not degraded.
These results are extraction process.
RNA extraction in the expected to be an alternative for laboratories field reported by Breitler et al.
can be with limited instruments in order to practice carried out at high temperatures .
Ae38AC) RNA isolation.
using a TRIzol reagent.
Abdallah et al.
also reported that the stability of MERS-CoV RNA on spin columns of RNA extraction kit Ina.
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: 73Ae80 Muhammad Taufiq Hidayat & Endry Nugroho Prasetyo conceptualization, resources, supervision.
CONCLUSIONS Based on the results of visualization of project administration, funding acquisition.
agarose gel on the UV transilluminator, we conclude that RNA isolation with the ACKNOWLADGEMENTS PureLinkA RNA Mini Kit can be carried out This study was supported by the Ministry in a laboratory with basic instruments such as of Research, and Technology / National a hand homogenizer and centrifuge without Research temperature control.
(RISTEKBRIN).
Republic of Indonesia, and Innovation Agency Institut Teknologi Sepuluh Nopember (ITS).
AUTHOR CONTRIBUTIONS Muhammad Taufiq Surabaya.
Indonesia under a grant number of Hidayat:
3/AMD/E1/KP.
PTNBH/2020
1177/PKS/ITS/2019.
analysis, investigation, writing - original CONFLICT OF INTEREST Endry Nugroho Prasetyo:
There are no conflicts of interest.
REFERENCES