INTRODUCTION:

Recently cancer research has revealed that RNA isolation from peripheral blood samples might provide essential and helpful information about the gene expression profile of tumor cells in the case of detecting the micro RNA or messenger RNA that are free in the blood stream[1].Moreover, isolated mRNA from white blood cells, may also give important knowledge about the transcriptome profile of immune cells and the influence of that on metastasis incidence and tumor progression[2-4].

Ribonucleic Acid (RNA) isolation from different types of samples is widely considered as a real challenge since the RNA molecule is an unstable, single stranded one that can be degraded easily. Furthermore, the RNase, an enzyme that degrades RNA, is a very stable enzyme that exists in the surrounding environment, resists most sterilization methods and causes degradation of the RNA[5]. Therefore, working with RNA requires being aware of certain precautions during isolation steps in order to protect the RNA from degradation as much as possible[6].

SUBJECTS AND METHODS:

RNA Isolation:

RNA was isolated from 30 peripheral blood samples that have been collected using EDTA anticoagulative tubes. Two RNA isolation methods were compared in order to find the most effective one, the first method was conducted using commercialRNA purification kit, this one depends on using specific columns (purification columns), that are supported with silica membrane on each column[7].Under certain conditions , including the presence of Chaotropic Salts (such as guanidine thiocyanate) and alcohol, the RNA binds to the silica membrane that can be dissolved using nuclease free water in later steps. Chaotropic salts have many roles in RNA isolation[8], beside their role in lysing the cells and protecting the RNA from degradation as they inhibit the RNase[9], they also neutralize the negative charges in the RNA structure in order to enhance its attraction and binding to the silica membrane in the presence of alcohols [10, 11].

The kit’s protocol was performed as follows, after red blood cells lysing in the 1.5ml blood sample tubes, the Lysis buffer (riches in Chaotropic salts) was added, with consideration that each 1ml of lysis buffer required 20μl of β-Mercaptoethanol[8, 12]. Absolute ethanol was then added to the sample, mixed well and the supernatant was transferred gradually to the purification column that was fixed to a collection tube (associated with the kit) with a centrifugation step after each addition of the supernatant at room temperature in 12000rpm for 1 min. The collected liquid in the collection tube was thrown away after each centrifugation step, then fixed again to the purification column, and followed by adding the Wash Buffer1, the previous step was repeated and the collected liquid in the collection tube was thrown away and fixed again to the purification column. After that 2 steps of washing using the Wash Buffer, a new clean 1.5ml tube was then fixed to the purification column and nuclease free water was added to dissolve and unbind the RNA from the silica membrane, it was then left about 50 seconds and centrifuged in 12000rpm for 1 min. The purification column was then thrown away and the 1.5ml tube containing the dissolved RNA was kept in -20°C, depending on the forward work.

The other method in RNA isolation is the conventional one, using the acidic phenol, which is a liquid/liquid extraction method that depends on separating a mixture of molecules according to their solubility in unmixed liquids[13, 14]. In acidic environment (pH= 4.4- 4.7) the extra protons (H+) in the medium neutralize the negative charges in the phosphate backbone structure of the double stand DNA helix. Therefore, DNA molecule becomes uncharged and transferred to the organic phase, whereas the single stranded RNA molecule remains soluble in the aqueous phase, as RNA is more acidic because the pKa of its groups is greater than that of the DNA[15, 16]. Proteins and lipids are transferred to the organic phase, beside that a proportion of proteins is located in the separating layer of the two phases[17, 18]. In the purpose of getting the most effective protocol in isolating intact RNA, many conditions have been optimized in the acidic phenol method.

Figure 1: illustrates the formation of three layers (aqueous, intermediate and organic) after the addition of the organic mixture to the polar aqueous phase. Nucleic acids (RNA and DNA) and proteins are distributed differently in the appropriate phase according to their solubility in the mediums.

First, the whole blood sample was centrifuged in 800rpm for 5 minutes to remove the supernatant plasma phase, with attention and care not to get close to the puffy coat, which contains the white blood cells, in the separating layer. Then, red blood cells were lysed and removed, many low isotonic solutions formulas were manually prepared and tried for that purpose (table-1). The supernatant, which has been formed after centrifugation of the sample in 2000 rpm for 10 minutes, was discharged and the white blood cells pellet at the bottom of the tube was used for the further extraction steps. The next step is to lyse the white blood cells that contain nucleus and nucleic acids, a number of different lysis buffer formulas were prepared in the laboratory and experimented in the studied blood samples (table-1).

In the acidic phenol method and after treating the samples with the suitable solutions (red blood cells) (table-1). The sample was incubated with 500μl WBC lysis buffer for one minute in a1.5ml tube, then an organic mixture, which is composed of acidic phenol: chloroform: Isoamyl alcohol, was added to the aqueous solute in an equal volume (table-1). The tube’s components were mixed well by vortex for one minute, and incubated in ice for 15 minutes, in order to give the chance of the protons to neutralize the negative charges in the DNA structure to be able to be transferred to the organic phase.RNA molecules sensitivity against temperature changes was considered by keeping the samples in ice during the isolation process to prevent RNA degradation as much as possible. After the incubation between the two phases (aqueous and organic), the sample was centrifuged in speed of 12000rpm for 15 minutes. The aqueous phase, which contains the RNA, was then pipetted and transferred to a new clean 1.5ml tube, with attention not to be closed to the separating layer between the two phases since that could cause protein and DNA contamination of the aqueous phase[19]. After that 1/10 of the aqueous phase’s volume was added from 3M sodium acetate. The extraction process was then repeated, in the purpose of purifying the aqueous phase from protein and DNA traces, by adding a volume of an organic mixture (acidic phenol 5: chloroform1) equal to the aqueous phase’s volume. The tube’s components were mixed well by vortex for 1 minute, incubated in ice for 15 minutes, and centrifuged in 12000rpm for 15 minutes. The aqueous phase that contains the RNA was then transferred carefully to a new clean 1.5ml tube. An additional step of adding chloroform to the aqueous phase was performed, in order to purify the aqueous layer from phenol traces that might be existed in it as well as to guarantee the removal of proteins from it. After that an equal volume of the aqueous phase was added from isopropanol 100% in the purpose of precipitating the RNA[20]. The aqueous phase was incubated with the isopropanol for 1 hour in -20ᶱC. The remaining amount of sodium acetate charges has a role in RNA participation by neutralizing the negative charges in the RNA structure in the presence of isopropanol alcohol that provides a suitable medium for alteration to be happened between Na+ and Po3- charges[13], that has made the RNA molecules insoluble in the aqueous phase and precipitated as a pellet at the bottom of the 1.5ml tube after centrifugation for 20 minutes in 12000rpm. The supernatant was then removed carefully and the RNA pellet was washed twice using ethanol 75%, to remove any trace of the used water soluble salts such as sodium acetate[20]. The eluted pellet was then centrifuged for 5 minutes in 4000rpm, the ethanol was thrown away and the tube was remained open for 15 minutes, to allow the ethanol to evaporate, but not leaving the tubes opened for a long time since that may cause dryness and degradation of the RNA pellet. After that the RNA pellet was then suspended using nuclease free water, mixed well using the vortex. The isolated RNA was then reserved in -20ᶱC[20].The table-1, illustrates a number of conditions that have been modified in the process in order to get the optimal ones for isolating intact RNA.

AGAROSE GEL ELECTROPHORESIS:

There are many ways to assist the efficacy of the isolated RNA[20], one of which is electrophoresis of the samples on agarose gel[21]. The results must observe two clear and sharp bands that refers to the ribosomal RNA 28S at 5000bp, and 18S at 2000bp compared to the ladder molecular weights, as well as the 28S band is twice in intensity as the 18S[22], the observation of the two bands previously mentioned indicates that the isolated RNA is intact. A number of agarose gel concentrations have been experimented, the agarose powder was mixed with TAE1X buffer, using microwaveable flask, then microwaved for 1-2 minutes until the agarose powder was dissolved completely, but with attention not to over boil to avoid buffer’s water evaporation. After the gel was cooled it poured in the gel slap and had been waited for 10 minutes in room temperature, then was transferred to 4+ᶱC fridge for 20 minutes until it was completely solidified. It had been noticed that preparing the agarose gel a day before performing the technique was better than applying the experiment immediately after the gel’s preparation.

The bubbles that might be contrasted during the gel preparation are preferred to be removed as they may disrupt the movement of the samples. The gel slap was then transferred to the electrophoresis pool and was overwhelmed with the TAE1X buffer. Using the DNA loading dye 6X(Thermo scientific®) that is composed of bromophenol blue and xylene cyanol FF, the samples were prepared to be applied in the wells. The loading dye was added in the ratio of 10 sample: 2 loading dye. In the purpose of comparing the results of the electrophoresed samples to known molecular weights, RNA Ladder (Thermo Scientific ®) had been used in the ratio of 6water:4Ladder: 2 loading dye. Optimisation of the electrophoresis optimal conditions had been tried in order to identify the best way to get the intact RNA, therefore many voltages intensities had been tried with modification of samples electrophoresis time. The staining step was performed after that, using Ethedium Bromide (0.5μg/μl), The results had then been assessed after the gel exposure to Ultra Violet waves.

RESULTS:

Assessing the quality of RNA isolation, requires the ribosomal RNA (18S, 28S) appearance as clear and sharp bands after electrophoresis of the RNA samples in agarose gel[23]. According to that, the results have illustrated that the acidic phenol is more effective as a RNA isolation method than the silica membrane one. A number of conditions have been modified (table-1), in order to get the perfect acidic phenol RNA isolation protocol. The results have revealed that the most efficient protocol that allows isolating intact RNA effectively was as follows: using a fresh peripheral blood sample, collected on EDTA anticoagulative tube, not more than 15 minutes from its collection. The red blood cells have been lysed completely and in short time relatively by using EDTA solution 0.1M.

The white blood cells were perfectly lysed using SDS containing lysis buffers specially SDS1%- 2% (table-1), however, the most effective lysis buffer that guarantees the completed lysing of the white blood cells as well as leading to intact RNA isolation, was the lysis buffer7 (table-1) that contains guanidine salts[15].The best organic mixture ratio in isolation was ( acidic phenol 125: chloroform 24: Isoamyl alcohol 1) in the first step of extraction followed by the second one that depends on using acidic phenol5: chloroform1. Moreover, the optimal pH degree of the acidic phenol was 4.4. It has also been revealed that adding an additional step in the extraction process using chloroform only in a volume equal to the aqueous one in order to remove protein and phenol traces from the aqueous phase was an effective purification step.

Figure 2: illustrates the results of RNA samples electrophoresis in 1.5% agarose gel, for 45 minutes , 60 volt, staining for 25 minutes using Ethedium Bromide 0.5μg/μl. Sample1: the RNA sample isolated using the lysis buffer 1 , sample 2: the RNA sample isolated using the lysis buffer2, sample3: the RNA sample isolated using the lysis buffer 3, Sample 4: the RNA sample isolated using the lysis buffer4. The four previous samples have been isolated by the acidic phenol method with the same process condition (applying the previous optimal mentioned condition with the only change of the WBC lysis buffer).

Figure 3: illustrates the results of RNA samples electrophoresis in 1.5% agarose gel, for 45 minutes, 60 volt, staining for 25 minutes using Ethedium Bromide 0.5μg/μl. Sample1: the RNA sample isolated using the lysis buffer 5, sample 2: the RNA sample isolated using the lysis buffer6, sample3: the RNA sample isolated using the lysis buffer 7, the three previous samples have been isolated by the acidic phenol method with the same process condition, (applying the previous optimal mentioned condition with the only change of the WBC lysis buffer). Sample 4: the RNA sample isolated using the silica membrane method.

Centrifugation of the samples at 4+ᶱC was dramatically better than doing the action at room temperature. In addition washing the RNA pellet twice using 75% ethanol, followed by centrifuging in 4000rpm, and suspending the RNA pellet in nuclease free water were the best conditions in order to get an intact and efficient RNA that can be used for other biological techniques. The electrophoresis is the way that had been used to assess RNA isolation efficacy, that revealed intact ribosomal RNA as well as messenger RNA smear (figure-1,2).

DISCUSSION:

The ribosomal RNA 18S, 28S, have been used as remarkable indicators for RNA isolation quality assessing. The 28S (5000bp) band must be two folds in intensity as 18S (2000bp). The smear of nucleic acids just above the 28S band indicates to the messenger RNA, however, the smear of the nucleic acids at the end of electrophoresis way, presents degraded RNA[22]. According to that, it has been shown that the acidic phenol method is better and more sensitive to small amounts of RNA than the silica membrane one as the later revealed no bands after RNA samples electrophoresis. The potential reasons behind that might be that the isolated RNA molecules were small, maybe because of degradation, and bound irreversibly to the silica membrane and cannot be eluted using the nuclease free water. Other possible reasons might be related to the sample size used in the process, however, alterations in that step have been done but no remarkable effects have been observed, therefore, the acidic phenol method was considered as the best method. In the acidic phenol method, EDTA 0.1M solution was the best one used in the purpose of removing and lysing red blood cells (RBCs) that do not contain nucleus and thus cannot be used in RNA isolation from the blood. EDTA has also a role in degrading cellular membranes[24], and the reason behind its selection as the best way to lyse RBCs is that by using it the lysing takes no more than 30seconds to be performed which is relatively shorter than the time that was required for completed lysing using the other RBCs lysis solutions that have been used. Moreover, the white blood cells (WBCs)lysis buffers that have been used and tried have revealed variant efficacy, and the most effective one was the lysis buffer7 that contains guanidine salts (table-1), since these salts not only lyse cellular membranes but also have many roles in RNA isolation including Rnase enzyme inhibition[9], therefore protecting the RNA from degradation and assure isolating intact RNA. The SDS containing lysis buffers have also revealed good quality in isolating intact RNA (figure-2)[25], which were better than Triton containing lysis buffers formulas. The more acidic pH degree (4.4) of the medium has participated in Rnase inhibition as well as the basic and major role in isolating only the RNA from the white blood cells. The organic mixture ratio (acidic phenol 125: chloroform 24: Isoamyl alcohol 1) has revealed its role in isolating the RNA effectively compared to the other ratio (table-1), in which the aqueous phase might have DNA and protein traces. The organic mixture components (the phenol, chloroform, and isoamyl alcohol) have major roles in solving the organic cellular components and separating them from the aqueous layer. In addition they play helpful role in proteins degradation such as the Rnase enzyme and therefore they participate in protecting the RNA from degradation[26]. Moreover the phenol keeps in its structure aqueous molecules that could lead to lose amounts of the aqueous phase and the isolated RNA, that’s why chloroform is added in order to reduce the polarity of the phenol and increase the lipophilic characteristics of the phenol in order to improve extraction quality and lead to sharper separation between the two phases[27, 28]. The isoamyl alcohol has a function in preventing foam formation in the organic mixture. Since the RNA molecules is very sensitive and can be degraded easily in the environment, some precaution must have been taken in order to protect them from degradation. The autoclave has been used to sterilise the extraction process tools (tips, tubes, the water used in solutions and buffers preparation). Moreover, using the 4+ centrifuge has also reduced the amount of degraded RNA dramatically. Washing the RNA pellet using 75% ethanol has been illustrated as a very important step in order to remove the extra water soluble salts (Na+) that might disrupt and inhibit the later biological processes. The RNA pellet suspended in nuclease free water must be saved in 20- C orless.

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Received on 14.07.2017 Modified on 10.08.2017

Research J. Pharm. and Tech 2017; 10(12): 4291-4296.

DOI: 10.5958/0974-360X.2017.00786.7

The variable component	The change that has been made
sample	· Peripheral blood samples used through 24 hours from collection · Peripheral blood sample used through one hour from collection · Fresh peripheral blood sample used through minutes from collection
Red blood cells lysis solutions	· NH4CL 1X solution,(0.01M NH4CL, 1mM NaHCO3, 0.1Mm EDTA) · TE 1X buffer, pH=8, (10mM Tris-HCL pH8.0, 1mM EDTA) · EDTA 0.1M solution
White blood cells lysis buffers	· LysisBuffer1, pH=7.4, (0.4M NaCl, 1M Tris-HCL pH=7.4, Triton 0.5%) · LysisBuffer2, pH=7.4, (0.4M NaCl, 1M Tris-HCL pH=7.4, Triton 0.75%) · LysisBuffer3, pH=7.3, (26mM EDTA, 10mM Tris-HCL, 0.5% SDS) · LysisBuffer4, pH=7.3, (52mM EDTA, 10mM Tris-HCL, 1% SDS) · LysisBuffer5, pH=6.8, (5mM EDTA pH=8, 50mM Tris-HCL pH=6.8, SDS 2%, Glycerol 5%, β Mercaptoethanol 1%) · LysisBuffer 6, pH=6.8, (50mM Tris-HCL pH=6.8, SDS 2%, Glycerol 5%, β Mercaptoethanol2%) · Lysis Buffer 7, Gene Jet RNA Purifiction Kit lysis buffer, (guanidine salts)
The organic mixture ratio	· Acidic Phenol 25: Chloroform 24: Isoamyl alcohol 1 · Acidic Phenol 125: Chloroform 24: Isoamyl alcohol 1
The pH degree of the phenol	· pH= 4.4 · pH= 4.7
The variable component	The change that has been made
The Centrifuge’s type	· Micro centrifuge that works at the room temperature · Micro centrifuge that works at 4+ temperature
RNA Pellet wash	· One time using 75% Ethanol · Two times using 75% Ethanol · Two times using 75% Ethanol, followed by one time wash using 95%Ethanol
Centrifugation speed after ethanol elution	· 12000rpm · 4000rpm
RNA solution	· Nuclease free water · TE1X Buffer