RNA Extraction from Coccolithophorids

CTAB Isolation of Genomic DNA from Coccolithophorids

 (adapted from the Short Protocols in Molecular Biology, Third Edition, 1995)

 Introduction

When isolating genomic DNA from coccolithophorids we employ two techniques commonly used when isolating genomic DNA: cetyltrimethylammonium bromide (CTAB) precipitation and cesium chloride gradient ultracentrifugation.  CTAB is a nonionic detergent that is frequently used when isolating DNA from polysaccharide rich tissues such as plant tissues. CTAB precipitation combined with a chloroform extraction is extremely effective for eliminating complex carbohydrates that can be problematic with other isolation procedures. We have determined previously that the CTAB-based protocol works very well for coccolithophorids. DNA may be further purified using a cesium chloride density gradient, but this is not absolutely necessary.

The single most difficult problem in DNA isolation is the degradation caused by endogenous nucleases.  Using the most common extraction methods, DNA is recovered in fragments of about 20 kbp; enzymes present in the tissue can quickly degrade the DNA into much smaller fragments.  For this reason, several precautions must be taken to reduce the activity of endogenous nucleases including selection of the appropriate tissues, keeping samples cold, denaturing proteins with concentrated salt solutions, using chelating agents to remove divalent cation cofactors required by nucleases, digesting proteins with protease, and denaturing proteins with organic solvents such as phenol and chloroform.  It is also important to use Molecular Biology grade chemicals and reagents, and nanopure water.  And all solutions should be autoclaved or filter sterilized. 

Coccolithophores are typically grown in batch cultures in artificial or filtered seawater at 18° C in an environmental growth chamber with a 12 light/dark cycle.  Cells are harvested during late log/early stationary phase and cell pellets are stored frozen at -20° C. Initially cells are resuspended in TE .  To lyse the cells a small amount of SDS and proteinase K are added, and the cells are incubated at 37° C.  The SDS in an anionic detergent that helps to break apart cell membranes while proteinase K is a protease that digests proteins and protects against DNAses. The nonionic detergent CTAB and NaCl are added to the cell lysate following the 37° C incubation to denature many of the proteins and help dissociate contaminants from the DNA. Chloroform/isoamyl alcohol extractions are used after the initial incubation to remove the denatured proteins and carbohydrates.   The nucleic acids complex with the CTAB in the aqueous phase and are precipitated using isopropanol.  The final stage of purification involves a cesium chloride gradient which separates the DNA and RNA and removes any remaining contaminants.  During the centrifugation the RNA and contaminating proteins pellet while the DNA settles at a position equivalent to its buoyant density.  SYBR green is used to visualize the DNA in the gradient.  The green dye binds to the major groove of DNA and fluoresces when placed under a UV light.  After the gradient has been centrifuged the DNA appears as a bright band in the tube.  The band can then be easily removed with a syringe needle.

Cesium chloride gradients yield large quantities of very high purity DNA.  After removing the dye and preciptating the DNA recovered from the gradient, the DNA is resuspended in the appropriate volume of TE Buffer and can be quantitated.  At this time the DNA is ready and can be used for restriction digests, cloning, southern blots, etc.

When high quality DNA is not required the cesium chloride gradient centrifugation steps can be eliminated.  The DNA obtained after the chloroform extraction and isopropanol extraction can be used for most purposes; restriction digests, southern blots, PCR amplification, etc.  It is important to note that to completely resuspend genomic DNA takes a significant amount of time.  We generally allow the DNA to incubate overnight and have found that spectrophotometric readings will increase dramatically by doing so.

1. Materials

   1. Chemicals and Reagents

      1. CTAB (Fisher)

      2. Tris (Fisher)

      3. EDTA (Fisher)

      4. NaCl (Fisher)

      5. Cesium Chloride (Fisher)

      6. SYBR Green I (invitrogen)

      7. Proteinase K (Promega)

      8. sodium dodecyl sulfate (SDS) (Fisher)

      9. Isopropanol (Fisher)

      10. Chloroform (Fisher)

      11. Isoamyl alcohol (Fisher)

      12. Ethanol (Fisher)

      13. Water bath  (VWR Scientific)

      14. Centrifuge (Beckman)

      15. Ultracentrifuge (Beckman)

      16. 18 guage needles (Fisher)

      17. Ultracentrifuge tubes (Beckman)

      18. Disposable 50 ml centrifuge tubes (Fisher)

   2. Solutions

      1. 5 M NaCl prepare with nanopure water and autoclave

      2. 10% SDS prepare with nanopure water and autoclave CTAB/NaCl Solution Dissolve 4.1 gm NaCl in 80 ml H2O and slowing add 10 gm CTAB (hexadecyltrimethyl ammonium bromide while heating and stirring.  It may be necessary to heat to 65° C.  Adjust to final volume of 100 ml and autoclave. 20 mg/ml proteinase K (stored in small aliquots at -20° C)

      3. 24:1 chloroform/isoamyl alcohol

      4. Isopropanol

      5. 70% ETOH prepared using DNase free water

      6. TE Buffer, 10 mM Tris, 1 mM EDTA, pH 7.5

    

2. Methods

   Note:  You will want to wear gloves and practice sterile technique to avoid introducing nucleases into your sample which will degrade the DNA.

    

   1. CTAB Isolation of Genomic DNA

      1. Grow 1 L cultures of E. huxleyi in artificial sea water to late log or arly stationary phase.  Harvest cells by centrifugation at 8,000 xg  for 10 minutes

      2. Resuspend the pellet in 9.5 ml of TE buffer.  Add 0.5 ml of 10% SDS and 50 ml of  20 mg/ml proteinase K.  Mix and incubate at 37° C for 1 hour

      3. Add 1.8 ml of 5 M NaCl and mix thoroughly.  Add 1.5 ml CTAB/NaCl solution, mix, and incubate 20 minutes at 65° C.

      4. Allow the sample to cool to room temperature before extracting with an equal volume of chloroform/isoamyl alcohol (24:1). Centrifuge for 10 minutes at 8,000 RPM to separate the phases. Remove the upper aqueous phase and SAVE

      5. Remove the upper aqueous phase and precipitate the nucleic acids by adding  2/3 volume cold isopropanol.  Mix and leave at -70° C for 30 minutes.

      6. Pellet the nucleic acids by centrifuging at 10,000 RPM for 10 minutes. Wash the pellet with 70% ethanol and allow the pellet to air dry for 5-10 minutes.

      7. Resuspend the pellet in 4 ml of TE.  Resuspend the pellet by gently swirling the tube.  To avoid shearing the DNA do not use a pipet to resuspend the pellet as the DNA is very fragile at this point.

       

   2. Cesium Chloride Gradient Centrifugation

      1. With the pellet in solution add 4.2 gms of CsCl and mix.

      2. Using a sterile pasteur pipet, pipet your sample into the ultracentrifuge tube.  Fill the tube clear up to the neck.  To do this, you may need to use some of the “filler solution” made by adding 1.041 gm CsCl/ml of TE.  Add 2 ul of 10,000X SYBR Green I (alternatively you can use ethidium bromide at 10 mg/ml)and then heat seal the tube.
         Note:  SYBR Green is a mutagen and should be handled only with gloves on.

      3. After sealing the ultracentrifuge tube, centrifuge at 52 K overnight.

      4. Remove the SYBR green stained DNA band using a syringe and an 18 gauge needle.  You will want to vent the tube first by tube prior to pulling the DNA band.  With the needle sitting in a disposable 15 ml tube, remove the barrel from the syringe and allow the DNA to drip from the needle by gravity.  Forcing the DNA out of the needle will shear the DNA.

      5. Extract the DNA several times with NaCl saturated isopropanol to remove the SYBR green.  After adding an equal volume of the isopropanol (upper layer), invert the tube several times, and then allow the phases to separate.  The SYBR green will move from the lower aqueous phase into the upper phase containing isopropanol.

      6. After removing the SYBR green, note the volume.  Precipitate the DNA by adding 2 volumes of sterile water.  Note the new volume, and add an additional 2 volumes (2X the new volume) of 100% ethanol.  Leave at -20° C overnight.

      7. Centrifuge at 10,000 xg for 10 minutes.  Decant the supernatant.  The DNA pellet should be a small white or translucent white pellet that is usually found in the depression area between the cylindrical and conical portions of the tube.

      8. Wash the pellet by adding 1 ml of 70% ETOH and transferring the sample to a microfuge tube.  Centrifuging at max speed for 5 minutes.  Decant the supernatant and wash again with 70% ETOH.

      9. Resuspend the pellet in 100 µl of sterile water or TE.

       

   3. Ultraviolet Spectroscopic Analysis of DNA

The absorbance measurement of nucleic acids at 260 nm is straightforward as long as any contributions from contaminants and buffer components are taken into account.  To eliminate such contributions, the absorption of the DNA sample is measured at different wavelengths to assess both the concentration and purity of a nucleic acid preparation.

A260 measurements are quantitative for relatively pure nucleic acid preparations.  While the absorbancy readings cannot discriminate between DNA and RNA, the ratio of absorbances at 260 and 280 nm can be used as an indicator of nucleic acid purity.  Proteins, for example, have a peak absorption at 280 nm that will reduce the A260/280 ratio.

 

Quantitation of DNA:

1. Prepare 500 ml of  a 1:100 dilution of the dissolved DNA by first adding 5 ml of  the concentrated solution to 495 ml of dH2O in a 1.7 ml microfuge tube.  Mix by vortexing.

2. Determine the absorbance of the 1:100 dilution on the spectrophotometer, using water as the blank.  Measure the absorbance of your sample at 260 nm and 280 nm.  If the A260 value is greater than 1.0, prepare and read the absorbance of a 1:1000 dilution.  If the A260 value is less than 0.1, prepare and read the absorbance of a 1:10 dilution.

3. The DNA concentration and the A260/A280 ratio will provide you with an estimation of both the concentration and the purity of your DNA sample.  The extinction coefficient for DNA is 50 mg/ml. In other words, when the A260 value is 1, if the pathlength is 1 cm,   then the concentration of DNA in solution is 50 mg/ml.  Pure DNA typically has an A260/A280 ratio between 1.7 and 1.9, depending on its base composition.

RNA Extraction from Coccolithophorids

(Adapted from Glick, B. R. and J. E. Thompson.  Methods in Plant Molecular Biology and Biotechnology.  CRC Press, Ann Arbor, Mich., 1993)

Introduction

In some respects, working with RNA is much more difficult than working with DNA or proteins, and in other respects it is much simpler.  The major concern when working with RNA is the activity of ribonucleases which are ubiquitous and exceedingly difficult to inactivate and/or get rid of.  Hence, considerable care must be taken to control ribonucleases.   Workers should always wear clean, disposable gloves and practice sterile technique to prevent ribonuclease contamination.  Glassware  is often baked at high temperatures (200° C) for several hours, and solutions are also often prepared with diethylpyrocarbonate (DEPC) treated water to avoid RNAses.  RNA, on the other hand, is easier to handle than DNA from the standpoint that it is tolerant of varied solvent, salt, and temperature conditions, and hence, denaturation is of little concern.  RNA is also highly resistant to shearing, and can be pipeted or vortexed without detriment.

When working with RNA it is important to define the appropriate tissue source and to determine the kind of isolation most appropriate for the experiments in mind.  For quantitative analyses and for most northern hybridizations total cellular RNA is adequate.  For cDNA synthesis or characterization of rare messages, however, polyadenylated RNA may be necessary.  Polysome-associated mRNA is required if one is interested in distinguishing actively translating mRNA.  If the focus is on organellar gene expression one may not want to isolate total RNA, but rather may want to initially purify the organelle, prior to isolating the RNA.

Although it is preferable to isolate total RNA from fresh cultures of coccolithophorids, total RNA can be isolated from cells that have harvested by centrifugation, rinsed in ethanol, centrifuged again, and stored at -20° C until the isolation can be performed.  For RNA extraction, cells are lysed by grinding with liquid nitrogen in a mortar and pestle.  The material is thawed in an extraction buffer containing guanidinium thiocyanate, a strong chaotropic agent that inhibits ribonucleases.  Sarkosyl and b-mercaptoethanol are also included in the extraction buffer.  Sarkosyl is added to both disrupt membranes and to inhibit ribonucleases while the b-mercaptoethanol is added to prevent free-radical dependent cross-linking of phenolics to DNA, and to reduce disulfide bonds present in ribonuclease molecules.  To separate RNA from other cellular components a phenol extraction, differential centrifugation, and multiple precipitations are performed.  A final precipitation with lithium chloride yields high quality RNA.

While the concentration and purity of RNA can be assessed using UV spectroscopy, it is essential to always check the integrity of the isolated RNA.  This is typically accomplished using a bioanalyzer or by aragrose gel electrophoresis.  When using agarose gel electrophoresis, because RNA is a single stranded molecule with its nitrogen bases exposed, it is capable of folding up on itself and forming extensive secondary structure.  The secondary structure of RNA molecules affects its migration through the gel.  In order to ensure that RNA migrates only with respect to molecular weight, samples of RNA must be denatured prior to, and during electrophoresis.  The most widely used RNA denaturants for agarose gel electrophoresis included formaldehyde and glyoxyl/dimethyl sulfoxide.  Formaldehyde gels typically afford greater detection sensitivity than the glyoxal denaturing system, and hence, in this investigation RNA electrophoresis will be performed using formaldehyde.  While formaldehyde is a carcinogen, it can be easily and safely managed in a fume hood.  RNA samples will be denatured before electrophoresis with formaldehyde and formamide.  Formaldehyde will also be added to the agarose gel to maintain the denatured state of the RNA during electrophoresis.  The running buffer used in conjunction with the formaldehyde gel is 1X MOPS/EDTA Buffer that is generally prepared as a 10X stock (0.2 M MOPS, pH 7.0, 50 mM sodium citrate, 10 mM EDTA, pH 8.0).  Formaldehyde gels are not as stiff as other agarose gels and are sometimes difficult to handle because they are so slick.

RNA electrophoresis is typically used to assess the integrity of isolated RNA.  A successful RNA extraction is indicated by the presence of two prominent ribosomal RNA (rRNA) bands with a faint smear of the different sized mRNA fragments visible in the background.  The absence of intact rRNA bands which represent 97% of the total RNA is indicative of degradation, which is almost certain to mean contaminating RNases are present.  Moreover, if the more prominent rRNA is degraded one can assume the small fraction of mRNA (representing only 3-5% of the total RNA) is degraded as well.

1. Materials

   1. Chemicals and Reagents

      1. Liquid Nitrogen

      2. Stainless Steel Mortar and Pestle (stainless steel is highly recommended and can be purchased from Fisher, catalog # 12-47-should be autoclaved before using.

      3. EDTA (Fisher)

      4. NaCl (Fisher)

      5. RNase free water (invitrogen)

      6. Guanidinium isothiocyanate (Fisher)

      7. Sodium citrate (Fisher)

      8. Sarkosyl (Fisher)

      9. β-mercaptoethanol (Fisher)

      10. Lithium Chloride (Fisher)

      11. Sodium Acetate (Fisher)

      12. SYBR Green I (invitrogen)

      13. Proteinase K (Promega)

      14. sodium dodecyl sulfate (SDS) (Fisher)

      15. Isopropanol (Fisher)

      16. Phenol pH 4.3 (Fisher)

      17. Chloroform (Fisher)

      18. Isoamyl alcohol (Fisher)

      19. Ethanol (Fisher)

      20. Water bath  (VWR Scientific)

      21. Centrifuge (Beckman)

      22. Ultracentrifuge (Beckman)

      23. 18 guage needles (Fisher)

      24. Ultracentrifuge tubes (Beckman)

      25. Disposable 50 ml centrifuge tubes (Fisher)

      26. RNAse Out (Ambion)

      27. HCL

      28. Sodium Hydroxide

      29. Disposable 5 ml and 10 ml pipets (Fisher)

      30. Vortex (Fisher)

      31. 37% Formaldehyde (Fisher)

      32. Formamide (Fisher)

      33. bromophenol blue (Fisher)

      34. ethidium bromide (Fisher)

   2. Solutions

      •  RNA Extraction Buffer (4 M guanidinium isothiocyanate, 25 mM sodium citrate, 0.5% sarkosyl, 0.1 β-mercaptoethanol) prepare using nanopure water and autoclave before adding the β-mercaptoethanol

      • 2 M Sodium acetate, pH 4.0, prepare using nanopure water and autoclave

      • 3 M Sodium acetate, pH 6.5, prepare using nanopure water and autoclave

      • 4 M Lithium Chloride, prepare using nanopure water and autoclave.

      • 0.1 MHCl prepare using autoclaved nanopure water

      • 0.1 M NaOH prepare using autoclaved nanopure water

      • 10X MOPS/EDTA Buffer (0.2 M MOPS, 50 mM sodium acetate, 10 mM EDTA, pH 7.0) will turn a golden color after autoclaving

      • 2X RNA Loading Buffer

        • 750 μl deionized formamide

        • 240 μl formaldehyde

        • 150 μl 10X MOPS/EDTA

        • 200 μl 50% glycerol

        • 10 μl 10 mg/ml ethidium bromide

        • 0.5 mg bromophenol blue

2. Methods

   Prepare the bench top and each of the pipetmen by wiping them down with RNAse Out.  Gloves must be worn at all times, and it is important to work quickly but carefully.  To avoid possible RNase contamination we use disposable pipets and centrifuge tubes as much as possible.

   1. Decalcification

      If one is working with cultures of calcifying cells, it is necessary to de-calcify the cells, otherwise the calcium carbonate will interefere with the extraction and subsequent enzymatic reactions.  Cultures can be decalcified by either treating them briefly with dilute acid, or by washing with EDTA.

      1. To 1 L cultures of calcifying cells add 22 ml of 0.1 M HCl to bring the final concentration to 2.2 mM.  Swirl the flask and note the clearing of the culture as the calcium carbonate liths begin to dissolve.  After adding the HCl, check to see that the pH has dropped from 8.0 to ~5.0 using pH strips.

      2. After 60 seconds, quickly neutralize the solution by adding 28.6 ml of 0.1 M NaOH (final concentration of 2.8 mM).  This serves to restore the culture to a neutral pH of 8.0. 

   2.  Harvesting Cells

      1. Cells are harvested by centrifugation at 10,000 xg for 10 minutes at room temperature or at the temperature of optimal cell growth.  After centrifugation do not place the cells on ice as this will cold shock the cells and activate specific RNAses within the cell.  Keep the cells at room temperature, or optimal growth temperature.

      2. If RNA is to be extracted at a later point in time, resuspend the cells in 100% ETOH and pellet the cells again by centrifugation before storing at -20˚.  If one is working with multiple bottles it is advantageous to combine cells before centrifugation, to arrive at one cell pellet preferably in a disposable 50 ml centrifuge tube. This may be accomplished by resuspending pellet from one bottle in 35 ml of ETOH, and transferring the cells to another bottle to resuspend the next pellet, and so on.

      3. If RNA is to be extracted immediately, and one has multiple bottles, to minimize the amount of material that is lost, resuspend and combine the cells before centrifuging, again to arrive at one cell pellet in a disposable 50 ml centrifuge tube

   3. RNA Extraction

      1. Transfer the cell pellet to a pre-chilled stainless steel mortor and grind coccolithophores in liquid nitrogen to a fine powder.  Transfer the cell material to a sterile 50 ml centrifuge tube.

      2. Add 10 ml of Extraction Buffer and vortex for 30 seconds.  Vent to release any pressure build-up

      3. Add 1 ml of 2 M sodium acetate, pH 4.0, and vortex for 30 seconds.

      4. Add 10 ml water-saturated phenol (pH 4.3) and vortex 30 seconds.

      5. Add 2 ml of chloroform:isoamyl alcohol (24:1) and vortex for 30 seconds.  Release any pressure build-up

      6. Centrifuge at room temperature for 10 minutes at 5000 xg to precipitate protein and    DNA.  Protein and DNA will form a thick interface between the aqueous and organic phases

      7. Remove the RNA containing upper aqueous phase and add an equal volume of  isopropanol.  Mix and incubate at -20° for at least 45 min (stopping point, can hold here a -20˚ C for an extended period of time).

      8. Pellet the RNA by centrifugation at 10,000 xg for 10 minutes at 4° C.

      9. Resuspend in 500 ml sterile water and transfer to a microfuge tube.  Add an equal volume of 4 M LiCl and mix.  Place on ice for 45 minutes.

      10. Centrifuge for 5 min at full speed.  Wash the pellet in cold 70% ethanol, air-dry, and resuspend in a small volume (50- 100 ml) of water.

      11. Determine the concentration of RNA from its absorbance at 260 nm.  At A260 an absorbance of 1 is equivalent to 40 ug/ml of RNA.  Pure RNA has an A260/A280 close to 2.0 and may be slightly higher.

   4. RNA electrophoresis

      1. Weight out sufficient agarose to make a 50 ml gel at 1.5% agarose. Generally a 1.5% agarose gel will provide separation of RNA molecules in the range of 0.5 to 2.5 kb.  A lower percentage agarose can be used for large RNA molecules while a higher percentage can be used for smaller RNAs.

      2. Add 36.5 ml of sterile water and 5 ml of 10X MOPS/EDTA Buffer.  Microwave until the agarose is dissolved.

      3. Allow the agarose to cool to about 55°C before adding 8.5 ml of 35% formaldehyde.  The formaldehyde should be added in the hood and handled with care as formaldehyde is a known carcinogen.  Swirl to thoroughly mix the formaldehyde with the agarose and immediately pour into the gel tray.

      4. Once the gel has solidified, place it into the electrophoresis unit and cover with 1X MOPS/EDTA Buffer.

      5. Prepare RNA samples by adding 2X RNA Sample-loading dye to bring the final volume to 1X.  Load 10-20 mg of E. huxleyi RNA per lane.  Load 2.5 mg of RNA size marker (each aliquot has 2.5 mg of of marker in 2.5 ml).

      6. Mix the samples well and heat at 65°C for 10 minutes prior to loading the gel.

      7. Load the samples into the wells of the gel and electrophoresis at 75 volts.

      8. When electrophoresis is complete, photograph the gel, and then rinse in sterile water with moderate agitation for 30 minutes.  Gels containing formaldehyde are less rigid than other agarose gels and are considerable more slippery.  Hence, it is wise to support the gels firmly when handling and/or when moving them from place to place.