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A general purpose method for extracting RNA from Dictyostelium cells

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A general purpose method for extracting RNA from Dictyostelium cells
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  See discussions, stats, and author profiles for this publication at: https://www.researchgate.net/publication/6290518 A general purpose method for extracting RNAfrom Dictyostelium cells  Article   in  Nature Protocol · February 2007 DOI: 10.1038/nprot.2007.191 · Source: PubMed CITATIONS 5 READS 48 5 authors , including:Pascale GaudetSwiss Institute of Bioinformatics 92   PUBLICATIONS   4,747   CITATIONS   SEE PROFILE Petra FeyNorthwestern University 63   PUBLICATIONS   2,987   CITATIONS   SEE PROFILE Rex L ChisholmNorthwestern University 148   PUBLICATIONS   7,655   CITATIONS   SEE PROFILE All content following this page was uploaded by Pascale Gaudet on 25 August 2014. The user has requested enhancement of the downloaded file. All in-text references underlined in blue are added to the srcinal documentand are linked to publications on ResearchGate, letting you access and read them immediately.   A general purpose method for extracting RNA from Dictyostelium  cells Karen E Pilcher 1 , Pascale Gaudet 1 , Petra Fey  1 , Anthony S Kowal 2 & Rex L Chisholm 1,2 1 dictyBase, Center for Genetic Medicine, Northwestern University, 676 North Saint Clair Street Suite 1260, Chicago, Illinois 60611, USA.  2 Center for Genetic Medicine,Northwestern University, 303 East Superior Room 7-125, Chicago, Illinois 60611, USA. Correspondence should be addressed to R.L.C. (r-chisholm@northwestern.edu). Published online 24 May 2007; doi:10.1038/nprot.2007.191 Here we present a protocol for the extraction of RNA from  Dictyostelium discoideum .  Dictyostelium  is a social amoeba that undergoesa basic developmental program, and therefore analysis of RNA levels over a time course is a commonly used technique. This procedureis similar to other guanidine thiocyanate-based methods; however, it has been adjusted because of the large quantities of carbohydrateand nucleases found in  Dictyostelium  cells. After cell lysis and phenol:chloroform extraction, the resulting high-quality RNA isolatedwith the described protocol allows the molecular genetic analysis of wild-type and genetically modified cells. The purified RNA can beused for analyses such as northern blotting, RT-PCR and microarrays. This procedure requires approximately 2 h to complete. INTRODUCTION D. discoideum  is a social amoeba that, upon starvation, aggregatesand forms a multicellular fruiting body  1 . This simple develop-mental program provides an excellent model for researchingdifferentiation and cell movement. In addition to developmentalmechanisms,  Dictyostelium  is well suited for studying processesinvolved in chemotaxis 2 and cell signaling 3 . Research is facilitatedby a complete genome sequence 4 that is housed and annotated atdictyBase 5 , the model organism database for  Dictyostelium . Dictyostelium  cells are easy to grow  6 and can be analyzedthroughout development for differential gene expression. A com-mon way to assay gene expression is through the isolation of RNAandsubsequentanalysisbynorthernblot 7 orbyRT-PCR  8 .Inrecent years, RNA has also been used for high-throughput experimentssuch as microarrays 9,10 . The extraction of RNA from  Dictyostelium isrelativelyeasy, asRNA levels are very high in comparisonto DNAlevels, estimated at approximately 40 times that of DNA 11 : 1  10 8 Dictyostelium  cells contain approximately  12 22  m g nuclear genomicDNA, 300  m g total RNA, 11 mg total cellular protein and 1.5 mgtotal carbohydrate. In this issue, we have also provided a generalpurpose protocol for extracting DNA 13 , as well as methods forculturing  Dictyostelium 6 and DNA transformation of cells 14 .The RNA isolation protocol presented here is adapted fromFranke  et al  . 15 and makes use of guanidine thiocyanate, a powerfulreagent that lyses cells and rapidly denatures proteins, includingribonucleases. The resulting RNA product is of high quality andcan be used for all downstream applications, including RT-PCR  8 and microarray experiments 9,10 . In our hands, we have successfully used this protocol to determine transcript levels by northernblot analysis 16–18 . Certain commercially available kits have beenused successfully; however, lysis conditions need to be adjustedowing to the high level of carbohydrates in  Dictyostelium  cells(http://dictybase.org/ListServ_archive/listserv_archive_molecbiol.html#genomic4-1). These kits include Trizol (Invitrogen; ref. 10) and RNeasy (Qiagen; ref. 19).RNA is a particularly labile molecule in all organisms, furthercomplicated in  Dictyostelium  because of the high level of degrada-tive enzymes, which support its lifestyle as a phagocyte 20 . Specialcare mustbe takenwhen extracting andhandling RNAto minimizeexposure ofthe sampleto nucleases. Thisincludesthe useofdiethylpyrocarbonate (DEPC)-treated water, RNA-grade phenol, cleangloves, barrier pipette tips, working on ice and careful attention tominimizing the amount of time for each step. MATERIALS REAGENTS . D. discoideum  (http://dictybase.org/StockCenter/StockCenter.html) . 10  KK2: 22 g KH 2 PO 4  (monobasic), 7.0 g K 2 HPO 4  (dibasic) in 1 liter;autoclave; dilute 1:10 with sterile distilled water for use at 1  concentration . GSEM buffer: 50% (w/v) guanidine thiocyanate, 0.5% (w/v) sarkosyl(Na-salt), 25 mM EDTA pH 8.0; adjust pH of GSEM buffer to 7.0; do notautoclave and store at 4  1 C; add 0.1% (v/v)  b -mercaptoethanol just beforeuse  !  CAUTION  Guanidine thiocyanate is hazardous; avoid inhalation andcontact with skin. . Phenol pH 4.7 (see REAGENT SETUP) . Chloroform  !  CAUTION  Chloroform is believed to be a carcinogen; avoidinhalation and use a fume hood while working with this substance. . Phenol:chloroform (1:1) pH 4.7 (see REAGENT SETUP) . DEPC-treated water: add 0.1% (v/v) DEPC to distilled water, shakevigorously, leave at room temperature (22  1 C) for 3 h to overnightand autoclave . 4 M NaOAc: adjust pH to 6.0 with acetic acid; use DEPC-treated water tomake solution in a baked bottle and autoclave . 100% (v/v) Ethanol  !  CAUTION  Ethanol is extremely flammable. . 70% (v/v) Ethanol m CRITICAL  Dilute 70% ethanol with DEPC-treatedwater. To minimize RNase contamination, always use fresh aliquots of ethanol or keep ethanol used for RNA preparations separate from reagentsused for other experiments. EQUIPMENT . Vortex  . Centrifuge . Spectrophotometer REAGENT SETUPPhenol pH 4.7  To 35 ml liquefied phenol, add 10 ml of 42 mM sodium citratepH 4.7. Shake vigorously and centrifuge at 2,500  g   for 10 min at roomtemperature. Remove the upper layer (sodium citrate), and repeat by adding10 ml sodium citrate, shaking, centrifuging and removing the sodium citratelayer. Then top the phenol off with 5 ml sodium citrate pH 4.7 and store at 4  1 Cin the dark (cover with aluminum foil if an opaque container is not available).Premixed phenol:chloroform pH 4.7 may be purchased (Ambion; cat. no.AM9720). m CRITICAL The pH of phenol is very important; an acidic pH    p  u  o  r   G   g  n   i   h  s   i   l   b  u   P   e  r  u   t  a   N    7   0   0   2   ©   n  a   t  u  r  e  p  r  o   t  o  c  o   l  s   /  m  o  c .  e  r  u   t  a  n .  w  w  w   /   /  :  p   t   t   h NATURE PROTOCOLS  |  VOL.2 NO.6  |  2007  |  1329 PROTOCOL  degrades DNA and thus partitions it to the organic phase while allowing theRNA to separate to the aqueous layer for subsequent extraction. It is highly recommended to use RNA-grade phenol to minimize possible exposure toRNases. Also, phenol oxidizes by a free radical process, indicated by apinkish color. Do not use oxidized phenol, as it can result in nucleicacid damage.  !  CAUTION  Phenol is caustic; always wear gloves and handlewith extreme care. Phenol:chloroform (1:1) pH 4.7  Mix an equal volume of phenol pH 4.7with chloroform. Store phenol:chloroform at 4  1 C in the dark (coverwith aluminum foil if an opaque container is not available). m CRITICAL Phenol:chloroformmustbewellmixed,andifstoredtogether,theywillseparate,so they must be mixed thoroughly again immediately before use.  !  CAUTION Phenol:chloroform is caustic; always wear gloves and handle carefully. It isrecommended to work in a fume hood while using this reagent. PROCEDURE1|  Grow and quantify  Dictyostelium  cells according to previously described methods 6 . 2|  Pellet 1  10 8 Dictyostelium  cells in a 50 ml polypropylene tube by centrifugation at 500  g   for 4 min at 4  1 C. Pour off thesupernatant. m CRITICAL STEP  It is important to wash the cells at a low temperature to make sure they do not change their gene expressionpatterns before lysis. This is particularly important if vegetative samples are required, as changes ingene expression may occur veryrapidly once nutrients are removed. 3|  Wash cells by adding 0.5 volume (25 ml) of cold KK2 buffer. Resuspend cells by vortexing and centrifuge at 500  g   for 4 minat 4  1 C. Pour off the supernatant. Repeat this step once. 4|  Resuspend cells in 1 ml cold KK2 buffer, transfer to a 1.5 ml microfuge tube and pellet in a microfuge by spinning at12,000  g   (highest setting for a typical benchtop microfuge) for 1 min at room temperature. Remove the supernatant using aP1000 micropipettor. m CRITICAL STEP  It is essential to carry out the remaining steps quickly and on ice to minimize degradation of the RNA. ’ PAUSE POINT   Cells can be snap-frozen in a dry ice–ethanol bath and stored at  80  1 C. Before use, thaw cells quickly withoutwarming them up significantly. Place on ice and continue with Step 5. 5|  Dissolve pellets in 200  m l GSEM buffer with vigorous vortexing for 1–2 min. m CRITICAL STEP  It is important to lyse cells thoroughly for optimal efficiency of RNA extraction, which might be difficult forcellsof later developmental stages. Vortex time can be increased to 5 min for post-aggregative cells to ensure proper lysis. 6|  Add 200  m l of phenol:chloroform pH 4.7 (1 volume) to separate the RNA from the DNA and proteins. Vortex for 30 s andthen centrifuge for 5 min at 12,000  g   at room temperature. Following centrifugation, two distinct phases should be seen: theRNA will be contained in the aqueous (upper) layer, whereas most of the proteins and genomic DNA will be in the organicphenol:chloroform (bottom) layer. An interphase containing denatured cellular material should be visible between the aqueousand organic layers. 7|  Remove the aqueous (upper) phase with a pipette and transfer to a clean 1.5 ml microfuge tube. m CRITICAL STEP  Avoid disturbing the interphase while removing the aqueous layer to prevent contamination of samples withproteins and other degraded material, which is undesirable for downstream use of the RNA and could potentially degrade the RNA. !  CAUTION  Discard the tube containing the used phenol:chloroform according to your institution’s hazardous waste guidelines. ?  TROUBLESHOOTING8|  Re-extract the RNA by adding 100  m l DEPC-treated water to the organic (bottom) phase, vortexing and centrifuging asdetailed in Step 6. This maximizes the RNA yield. 9|  Remove the aqueous (upper) layer from Step 8 and combine with that from Step 7. 10|  Extract the pooled aqueous layer twice with 300  m l (one volume) of phenol:chloroform as described in Steps 6 and 7. 11|  Extract twice with 300  m l (one volume) of chloroform, as in Steps 6 and 7 (omitting phenol). These final extractions withchloroform are intended to remove any remaining phenol, as it is inhibitory for downstream applications. 12|  Add 15  m l (1/20 volume) of 4 M NaOAc and 600  m l (2 volumes) of ice-cold 100% ethanol to precipitate the RNA. Leave onice or at  20  1 C for at least 10 min. ’ PAUSE POINT   The RNA can be left in ethanol at  20  1 C overnight or at  80  1 C for several days. ?  TROUBLESHOOTING13|  Centrifuge for 10 min at 12,000  g   at room temperature in a microfuge. Discard the supernatant (ethanol) with a pipette. m CRITICAL STEP  Remove the ethanol carefully, taking care not to touch the RNA pellet. RNA pellets exist as large streaks that runup the side of the tube. Compared to DNA pellets, which tend to form globs at the bottom of the tube, they are not very compact.    p  u  o  r   G   g  n   i   h  s   i   l   b  u   P   e  r  u   t  a   N    7   0   0   2   ©   n  a   t  u  r  e  p  r  o   t  o  c  o   l  s   /  m  o  c .  e  r  u   t  a  n .  w  w  w   /   /  :  p   t   t   h 1330  |  VOL.2 NO.6  |  2007  |  NATURE PROTOCOLS PROTOCOL  14|  Rinse the RNA pellet twice by adding 70% ice-cold ethanol, followed by centrifugation for 1–2 min at 4  1 C. Pipetteoff the supernatant. Similar to the previous step, care should be taken to not disturb the RNA pellet when removingthe ethanol. 15|  Let pellet dry at room temperature for approximately 15 min and dissolve in 20  m l DEPC-treated water. Note thatRNA does not dissolve easily. Leave samples on ice for 15–30 min to rehydrate and then resuspend by pipetting upand down. m CRITICAL STEP  Do not let the RNA pellet dry too long, as it makes dissolving the pellet in water a difficult task. ’ PAUSE POINT   RNA samples are stable for several years at  80  1 C in DEPC-treated water. ?  TROUBLESHOOTING16|  Determine the RNA concentration by measuring the optical density (OD). Dilute RNA 1:1,000 (serial dilutions are highlyrecommended) in DEPC-treated water and determine the OD 260  using a spectrophotometer. Calculate the RNA concentrationwith the following formula: OD 260  reading  dilution factor   ((40  m g ml   1 )/OD 260  unit) ¼ RNA concentration ( m g ml   1 ).To calculate the RNA concentration in  m g  m l   1 , divide by 1,000. To determine the quality of the resulting RNA, take an OD 280 reading, which is the optimum absorption for proteins. Pure RNA has an OD 260  /OD 280  ratio in the range of 1.8–2.0. OD 260  /OD 280 ratios below 1.8 indicate contamination with proteins and/or phenol. ?  TROUBLESHOOTING  TIMING This protocol takes approximately 2 h to complete, from start to finish. ?  TROUBLESHOOTING Troubleshooting advice can be found in  Table 1 .    p  u  o  r   G   g  n   i   h  s   i   l   b  u   P   e  r  u   t  a   N    7   0   0   2   ©   n  a   t  u  r  e  p  r  o   t  o  c  o   l  s   /  m  o  c .  e  r  u   t  a  n .  w  w  w   /   /  :  p   t   t   h TABLE 1  |  Troubleshooting table. Step Problem Solution 7 RNAsampledoes not work indownstreamapplications,for example, RT-PCRSome contaminants may have been pulled out of the interphasewhen the aqueous layer was removed. Residual proteins and other cellular matter can inhibit enzymes. Be conservative when pipettingoff the aqueous layer, leaving a small amount near the interphase12 No precipitate is visible in the tube Leave on ice for an additional 5–10 min and check again or leave at  20  1 C for several hours15 RNA does not dissolve easily, observed either whilemanually resuspending or as seen in uneven loading of RNA on a gel Insteadof resuspending the RNA pellet inDEPC-treatedwater, use TEpH 8.0 (made with DEPC-treated water)16 RNA is degraded, as evidenced by a smear on theagarose gel RNases (ribonucleases), which rapidly degrade RNA, may be presentin the sample. Be sure to always wear gloves and use DEPC-treatedwater to minimize the risk of contamination. Working with samplesin a laminar flow hood and using barrier tips on your micropipettor can also help to reduce contaminationNo RNA is present in the end sample The pellet might have been lost during the washes with ethanol. Becareful not to disturb the pellet, which should be easily visibleOD 260  reading is greater than 1.0 OD readings greater than 1.0 are unreliable. Dilute the RNA samplefurther, and be sure to change the dilution factor in your calculationof the RNA concentrationOD 260  reading is less than 0.1 OD readings less than 0.1 are unreliable. Dilute the RNA sample witha factor of less than 1,000. To minimize loss of the RNA sample, it isrecommended to initially perform serial dilutions to reach a 1:1,000dilution. One of the serial dilutions can then be used to determinetheOD 260 .Besuretochangethedilutionfactorinyourcalculationof the RNA concentration NATURE PROTOCOLS  |  VOL.2 NO.6  |  2007  |  1331 PROTOCOL  ANTICIPATED RESULTS This protocol should yield approximately 300  m g of RNA from1  10 8 cells, or 15  m g  m l   1 when dissolved in 20  m l of DEPC-treated water. The extracted RNA should be of high quality,with an OD 260  /OD 280  ratio of 1.8 or above.  Figure 1  shows 0.5 m g of RNA in each lane on a 1% non-denaturing agarose gel,which results in tight bands, as seen here. Prominent bandsrepresenting the 26S (top band) and 17S (bottom band)ribosomal RNAs are an indication of good RNA. If these bandsare not present, the RNA sample is degraded and should notbe used for further applications. When RNA is run on adenaturing MOPS gel (3-(N-morpholino)propanesulfonic acid;see Fourney  et al  . 7 ), bands will be slightly diffuse and a faintstreak of RNA (representing different transcript sizes) will appear in the background of the lane. A MOPS gel is necessaryfor northern blotting to fully separate transcripts of differentsizes and for probe hybridization to occur. ACKNOWLEDGMENTS  dictyBase (http://www.dictybase.org) is supported by grantsfrom the NIH (GM64426 and HG00022). COMPETING INTERESTS STATEMENT   The authors declare no competing financial interests.Published online at http://www.natureprotocols.comRights and permissions information is available online at http://npg.nature.com/ reprintsandpermissions1. Chisholm, R.L. & Firtel, R.A. Insights into morphogenesis from a simpledevelopmental system.  Nat. Rev. Mol. Cell Biol.  5 , 531–541 (2004).2. Firtel, R.A. & Chung, C.Y. The molecular genetics of chemotaxis: sensing andresponding to chemoattractant gradients.  BioEssays  22 , 603–615 (2000).3. Mahadeo, D.C. & Parent, C.A. Signal relay during the life cycle of   Dictyostelium . Curr. Top. Dev. Biol.  73 , 115–140 (2006).4. Eichinger, L.  et al.  The genome of the social amoeba  Dictyostelium discoideum . Nature  435 , 43–57 (2005).5. 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Nellen, W.  et al.  Molecular biology in  Dictyostelium : tools and applications.in  Methods in Cell Biology. Volume 28: Dictyostelium discoideum: Molecular  Approaches to Cell Biology   (ed. J.A. Spudich) 67–100 (Academic Press Inc.,Orlando, FL, 1987).12. Ashworth,J.M.& Watts,D.J.Metabolismofthecellularslimemould Dictyosteliumdiscoideum  grown in axenic culture.  Biochem. J.  119 , 175–182 (1970).13. Pilcher, K.E., Fey, P., Gaudet, P., Kowal, A.S. & Chisholm, R.L. A reliable general purpose method for extracting genomic DNA from  Dictyostelium  cells. Nat. Protoc.  2 , 1325–1328 (2007).14. Gaudet, P., Pilcher, K.E., Fey, P. & Chisholm, R.L. Transformation of  Dictyostelium discoideum  with plasmid DNA.  Nat. Protoc.  2 , 1317–1324(2007).15. Franke, J., Podgorski, G.J. & Kessin, R.H. The expression of two transcripts of thephosphodiesterase gene during the development of   Dictyostelium discoideum . Dev. Biol.  124 , 504–511 (1987).16. Gaudet, P., MacWilliams, H. & Tsang, A. Inducible expression of exogenous genesin  Dictyostelium discoideum  using the ribonucleotide reductase promoter.  Nucleic  Acids Res.  29 , e5 (2001).17. Gaudet, P. & Tsang, A. Regulation of the ribonucleotide reductase small subunitgene byDNA-damaging agents in  Dictyostelium discoideum .  Nucleic Acids Res. 27 ,3042–3048 (1999).18. Pollenz, R.S., Chen, T.L., Trivinos-Lagos, L. & Chisholm, R.L. The  Dictyostelium essential light chain is required for myosin function.  Cell   69 , 951–962 (1992).19. Winckler, T.  et al.  CbfA, the C-Module DNA-binding factor, plays an essential role in the initiation of   Dictyostelium discoideum  development.  Eukaryot. Cell   3 ,1349–1358 (2004).20. Cardelli, J.A., Golumbeski, G.S., Woychik, N.A., Ebert, D.L., Mierendorf, R.C. &Dimond, R.L. Defining the intracellular localization pathways followed bylysosomal enzymes in  Dictyostelium discoideum . in  Methods in Cell Biology.Volume 28: Dictyostelium discoideum: Molecular Approaches to Cell Biology  (ed. J.A. Spudich) 139–155 (Academic Press Inc., Orlando, FL, 1987).    p  u  o  r   G   g  n   i   h  s   i   l   b  u   P   e  r  u   t  a   N    7   0   0   2   ©   n  a   t  u  r  e  p  r  o   t  o  c  o   l  s   /  m  o  c .  e  r  u   t  a  n .  w  w  w   /   /  :  p   t   t   h 124.1 kb1.9 kb Figure 1  |  RNA isolated from  D. discoideum . A 0.5  m g portion of RNA was runin each lane on a 1% non-denaturing agarose gel. Lane 1 contains RNA isolatedfrom strain AX3 grown on plates, whereas lane 2 contains RNA from AX3 cellsgrown in suspension. The upper 4.1 kb band represents the 26S ribosomal RNA,whereas the lower 1.9 kb band represents the 17S ribosomal RNA. 1332  |  VOL.2 NO.6  |  2007  |  NATURE PROTOCOLS PROTOCOL View publication statsView publication stats
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