Optimization of Total RNA Extraction Methods

Total RNA was isolated from B. rotunda tissues using different methods to obtain the optimized method, which fits B. rotunda at most efficiency. RNA sample must be free of RNase activity, any inhibitors of reverse transcription and PCR. RNase can be introduced at any point during the isolation procedure; therefore the required laboratory apparatus were designated for RNA work to prevent cross contamination. The bottles and pipette tips were soaked overnight in 0.1% (v/v) DEPC water, autoclaved at 121°C for 45min and dried in Drying oven 37°C overnight. Disposable gloves were used as skin often contains bacteria and molds as a source of RNase. During RNA experiments, samples were devoid of RNase contamination and aseptic conditions were maintained.

B. rotunda tissues in all methods was ground in a mortar with a pestle using liquid nitrogen except for TRIzol® method. It is essential to use the correct amount of starting material in order to obtain optimal yield and purity for RNA, so a maximum amount of 100mg plant material was used in all the methods. RNA pellet can be dissolved in PCR compatible buffer or water.

TRIzol® Method 3.9.2.1

TRIzol® reagent is toxic if it is in contact with skin or swallowed. It also causes burns;

therefore extra caution was taken while working with TRIzol® and if it was contacted with skin, was immediately washed with plenty of detergent and water. The reagent has shown stability of twelve months when stored at room temperature however it was stored at 2 to 8°C for optimal performance. TRIzol® reagent is a ready to use reagent for isolation of total RNA from cells and tissues. A mono-phasic solution of phenol and guanidine isothiocyanate is an improvement to the single-step RNA isolation method developed by Chomczynski and Sacchi. In homogenization step, a 100mg of B. rotunda tissue was weighed using Setra EL-2000S, added to 1ml of TRIzol® reagent and

ground using ten beads of 0.5mm ZrSiO or 1mm ZrO in a homogenizer. The sample volume did not exceed 10% of the volume of TRIzol® reagent. TRIzol® reagent was added in a chemical fume hood and breathing the vapor was strongly avoided. The tissue was not thawed during disruption with TRIzol® reagent. Homogenized sample was incubated at 55°C for 10min at Water Bath, Memmert. During sample homogenization, TRIzol® reagent maintains RNA integrity, while disrupting cells and dissolving cell components. Since tuberous parts of the plant tissue e.g. rhizome and root are rich in polysaccharides, an additional isolation step was required to remove insoluble material; therefore the homogenate was centrifuged at 13000rpm for 10min at 4°C Centrifuge 5417 R, Eppendorf. The pellet contains extracellular membranes, polysaccharides, and high molecular weight DNA while the supernatant contains RNA.

The clear supernatant was transferred to a new tube.

At phase separation step, the homogenized sample was incubated for 5min at room temperature to permit complete dissociation of nucleoprotein complexes. A 0.2ml of chloroform per 1ml of TRIzol® reagent was added to the tubes, and vigorously mixed for 15sec. The tubes were then incubated at room temperature for 3min and centrifuged at 13000rpm for 15min. Chloroform separates the solution into an aqueous phase and an organic phase. In other words, the mixture was separated into a lower red, phenol-chloroform phase, an interphase, and a colorless upper aqueous phase where RNA exclusively remains. Volume of the aqueous phase was about 60% of volume of TRIzol® reagent used for homogenization (~600µl). The organic phase was discarded since isolation of DNA and proteins were not aim of the experiment.

For RNA precipitation step, the aqueous phase was transferred to a new tube, mixed with 0.5ml isopropyl alcohol per 1ml of TRIzol® reagent. The sample was incubated at room temperature for 10min and centrifuged at 13000rpm for 10min. The RNA

precipitate was often invisible before centrifugation, formed a gel-like pellet on the side and bottom of the tube.

For RNA washing step, the RNA pellet was washed with 1ml of 75% ethanol per 1ml of TRIzol® reagent. The sample was mixed by vortex in Mixer Uzusio VTX-3000L LMS and centrifuged at 10000rpm for 5min. The RNA pellet was briefly air dried for 5min as very dry pellet decreases the solubility. Centrifugation under vacuum was avoided for RNA pellet. The RNA pellet was dissolved in 30µl RNase-free water and analyzed on 1% agarose gel.

Cetyltrimethylammonium Bromide-NETS Method 3.9.2.2

Total RNA was isolated from B. rotunda tissues using a modified Cetyltrimethylammonium Bromide-NaCl:EDTA:Tris:SDS (CTAB-NETS) method by Kiefer, Heller and Ernst (2001). The 100mg tissue was ground in a mortar with a pestle using liquid nitrogen. The powder filled a 1.5ml microcentrifuge tube containing 1ml extraction buffer preheated at 65°C at Water Bath, Memmert and 20µl β-mercaproethanol. The tissue was not thawed during disruption in extraction buffer. The extraction buffer contains 2% CTAB, 2% PVP40, 100mM Tris-Cl pH 8.0 (PB-10 Sartorius pH Meter) 25mM EDTA, 2M NaCl, and 2% (v/v) β-ME, which was freshly added to the extraction buffer. The tissue was mixed by vortex in Mixer Uzusio VTX-3000L LMS. An equal volume (1ml) of chloroform:isoamyl alcohol (24:1 v/v) was added and mixed by vortex. The mixture was centrifuged at 13000rpm for 15min at room temperature at Centrifuge 5415 D, Eppendorf and the supernatant was transferred to a new 1.5ml microcentrifuge tube. This step was repeated twice until the middle layer was clear of any proteins impurity. A 270µl of 7.5M LiCl was added to the supernatant and mixed using Mixer Uzusio VTX-3000L LMS. The mixture was incubated overnight at 4°C.

The sample was kept on ice during the experiment and centrifuged at 10000rpm for 30min at 4°C at Centrifuge 5417 R, Eppendorf. The supernatant containing DNA and other cell debries was discarded. A 500µl fresh-prepared NETS was added to the sample. A 20ml NETS solution contains 1M NaCl, 10mM Tris-Cl, 1mM EDTA, and 5% SDS. A 500µl of chloroform:isoamyl alcohol (24:1 v/v) was then added to the sample and centrifuged at 10000rpm for 25min at 4°C. The pellet containing DNA and other cell debries were discarded and the supernatant was transferred to a new tube with 0.1 volume of 3M NaOAc pH 5.2 (about 100µl) and 3 volume of ice-cold (4°C) absolute ethanol (about 1ml). The sample was incubated overnight at -80°C to precipitate RNA.

The sample was centrifuged at 4°C for 30min at 10000rpm. The supernatant was discarded and the pellet was washed with 1ml ice-cold 70% ethanol, centrifuged at 10000rpm for 5min at 4°C. The supernatant was discarded and the pellet was dried by heating at 50°C in a Dry Bath Incubator Major Science (MS) for 10min. The pellet was dissolved in 20-50µl DEPC-treated dH2O depending on size of the pellet. The extracted RNA was stored at -20°C for short-term usage (1-2 days) and at -80°C for long-term usage (up to one year). The RNA sample was analyzed on 1% agarose gel.

Cetyltrimethylammonium Bromide Method 3.9.2.3

Total RNA was isolated from B. rotunda tissues using a modified Cetyltrimethylammonium Bromide (CTAB) method by Kiefer, Heller and Ernst (2000).

The 100mg tissue was ground in a mortar with a pestle using liquid nitrogen. The powder filled a 1.5ml microcentrifuge tube containing 1ml extraction buffer and 20µl β-mercaproethanol. The tissue was not thawed during disruption in extraction buffer.

The extraction buffer contains 2% CTAB, 2% PVP40, 1M Tris Cl pH 8.0 (PB-10

added to the extraction buffer. The tissue was vortex using Mixer Uzusio VTX-3000L LMS. An equal volume (1ml) of different washing solutions was added to each sample to optimize the washing step. 1): Twice with PC (1:1), 2): Once with phenol and once with PC (1:1), 3): Once with PCIA (125:24:1) and once with CIA (24:1), 4): Once with PC (1:1) and once with CIA (24:1), 5): Twice with CIA (24:1). All samples were centrifuged at 13,000rpm for 15min at room temperature and the supernatant was transferred to a new 1.5ml microcentrifuge tube.

A 0.1 volume of 3M NaOAc pH 5.2 or 7.5M LiCl (about 100µl) and 3 volume of ice-cold (4°C) absolute ethanol (about 1ml) were added to the sample and incubated overnight at -80°C to precipitate RNA. The sample was kept on ice during the experiment and centrifuged at 4°C for 30min at 10000rpm in Centrifuge 5417 R, Eppendorf. The supernatant was discarded and the pellet was washed with 1ml ice-cold 70% ethanol and centrifuged at 10000rpm for 5min at 4°C. The supernatant was discarded, the pellet was air dried by inverting the tube for 5min and dissolved in 20-50µl DEPC-treated dH2O depending on size of the pellet. The RNA sample was analyzed on 1% agarose gel.

RNA Isolation Kit 3.9.2.4

Total RNA was isolated using RNeasy Mini Kit, Qaigen at room temperature. The 100mg tissue was immediately placed in liquid nitrogen, and thoroughly ground with a mortar and pestle. The tissue powder was transferred to RNase-free, liquid nitrogen–

cold, 1.5ml microcentrifuge tube while the liquid nitrogen was allowed to evaporate. A 450µl Buffer RLT containing 5µl β-ME was added to the powder in a fume hood and vigorously vortex using Mixer Uzusio VTX-3000L LMS.

The sample was incubated for 3min at 56°C in order to disrupt the tissue. The RNeasy Plant Mini Kit provides a choice of lysis buffers: Buffer RLT and Buffer RLC, which

contain guanidine thiocyanate and guanidine hydrochloride, respectively. In most cases, Buffer RLT is the lysis buffer of choice due to the greater cell disruption and denaturation properties of guanidine thiocyanate. Sometimes, guanidine thiocyanate can cause solidification of the sample, making RNA extraction impossible; therefore Buffer RLC is recommended. The sample was not incubated at high temperatures to avoid swelling of the sample and RNA degradation. The lysate was transferred to a QIAshredder spin column placed in a 2ml collection tube and centrifuged for 2 min at full speed. The RNeasy spin column was not overloaded, as this significantly reduces yield and quality of RNA. All centrifugation steps were performed at 20–25°C in a standard microcentrifuge.

The supernatant was transferred to a new microcentrifuge tube without disturbing the cell debris in the collection tube. The end of the pipet tip was sometimes cut off to facilitate pipetting of the lysate into the QIAshredder spin column. Centrifugation through the QIAshredder spin column removes cell debris and simultaneously homogenizes the lysate. Most of the cell debris was retained on the QIAshredder spin column and a very small amount of cell debris passed through and formed a pellet in the collection tube. This pellet was not disturbed when transferring the lysate to the new microcentrifuge tube. A 0.5 volume of absolute ethanol was added to the clear lysate, and mixed by pipetting. Precipitation after addition of ethanol did not affect the procedure. The 650µl sample including any precipitate was transferred to an RNeasy spin column placed in a 2ml collection tube and centrifuged for 15sec at 13000rpm using Eppendorf Minispin®. The flow-through was discarded and the collection tube was reused in next step. Sometimes the sample volume exceeded 700µl, thus successive aliquots were centrifuged in the same RNeasy spin column and the flow-through was discarded after each centrifugation. A 700µl Buffer RW1 was added to the RNeasy spin

13000rpm to wash the spin column membrane. The flow-through was discarded and the collection tube was reused in next step. After centrifugation, the RNeasy spin column was carefully removed from the collection tube so that the column was not in contact with the flow-through. A 500µl Buffer RPE was added to the RNeasy spin column, the lid was closed gently and the sample was centrifuged for 15sec at 13000rpm. The flow-through was discarded. This step was repeated twice and the collection tube was reused.

The long centrifugation dried the spin column membrane, ensuring that no ethanol was carried over during RNA elution. Residual ethanol may interfere with downstream reactions. Buffer RPE was supplied as a concentrate, thus absolute ethanol was added to Buffer RPE as indicated on the bottle to obtain a working solution.

After centrifugation, the RNeasy spin column was carefully removed from the collection tube so that the column was not in contact with the flow-through. Otherwise, carryover of ethanol was occurred. The RNeasy spin column was then placed in a new 2ml collection tube, the lid was closed gently and the sample was centrifuged at full speed for 1min. This step was performed to eliminate any possible carryover of Buffer RPE or flow-through on the outside of the RNeasy spin column.

The RNeasy spin column was then placed in a new 1.5ml collection tube and 30µl RNase-free water was added directly to the spin column membrane. The lid was closed gently and the sample was centrifuged for 1min at 13000rpm to elute the RNA. This step was repeated with additional 30µl RNase-free water to elute leftover RNA in the column. The RNA sample was analyzed on 1% agarose gel.

Gel Extraction Method 3.9.2.5

Gel extraction method was used to excise total RNA without DNA contamination. A 10µl of RNA isolated sample using modified CTAB method was loaded on 2% agarose gel and run for 25min at 120V using Consort EV265 Electrophoresis Power Supply.

The RNA parts on the gel were then excised under UV in a dark room on Vilber Lourmat Transilluminator and transferred to a 1.5ml tube containing 1ml 0.5M NaCl and incubated on ice for 4hrs on a Rocky 3D Belly Dancer with the speed of 3. The incubation was continued overnight at 4°C. The aqueous part was transferred to a new tube where 0.1 volume of 3M NaOAc pH 5.2 (about 100µl) and 3 volume of ice-cold (4°C) absolute ethanol (about 1ml) were added and incubated overnight at -80°C to precipitate RNA.

The sample was kept on ice during the experiment and centrifuged at 4°C for 30min at 10000rpm in Centrifuge 5417 R, Eppendorf. The supernatant was discarded and the pellet was washed with 1ml cold 70% ethanol and centrifuged at 10000rpm for 5min at 4°C. The supernatant then was discarded, the pellet was air dried by inverting the tube for 5min and dissolved in 20-50µl DEPC-treated dH2O depending on size of the pellet.

The RNA sample was analyzed on 1% agarose gel.