RNA Extraction From Cartilage 101

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From: Methods in Molecular Medicine, Vol. 100: Cartilage and Osteoarthritis, Vol. 1: Cellular and Molecular ToolsEdited by: M. Sabatini, P. Pastoureau, and F. De Ceuninck © Humana Press Inc., Totowa, NJ

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RNA Extraction From Cartilage

Frédéric Mallein-Gerin and Jérôme Gouttenoire

SummaryThe direct isolation of RNA from cartilage has often proved difficult owing to a number of

factors. Cartilage has a low cell content and contains an extracellular matrix rich in proteoglycans,which copurify with the RNA as they are large and negatively charged macromolecules. In ourlaboratory, we are interested in searching for genes differentially expressed in chondrocytes indiverse in vivo situations, for instance during maturation of chondrocytes in the growth plate orduring cartilage degeneration. We found that treatment by proteinase K in 1 M guanidiniumisothiocyanate prior to cesium trifluoroacetate ultracentrifugation was crucial to increase the yieldand purity of RNA extracted from cartilage matrix. This protocol indeed led to reproducible pat-terns of differential display reverse transcriptase-polymerase chain reaction (RT-PCR) and shouldbe useful for identifying genes differentially expressed by chondrocytes in situ.

Key Words: RNA; extraction; cartilage; chondrocyte; RT-PCR.

1. IntroductionThe use of molecular biology techniques has helped elucidate the metabolism

of normal and many pathological processes. Application of these techniques tohuman adult articular cartilage has been hampered by a number of factors. Carti-lage has a low cell content and a highly crosslinked extracellular matrix contain-ing a high concentration of proteoglycans entrapped in a collagenous network.Separation of RNA from the aggregating proteoglycans poses a problem becauseboth classes of molecules are extremely large and highly negatively charged.Therefore, most gene expression studies are based on RNA extracted from cul-tured chondrocytes. In situ hybridization can represent an alternative techniquefor studying chondrocyte gene expression in cartilage, but this technique is notquantitative. In our laboratory, we are interested in studying gene expressionduring cartilage development or progression of osteoarthritis in human and dif-ferent animal models. We describe here a reliable protocol, developed by modi-

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fying existing procedures, to isolate DNA-free RNA from cartilage. Purified totalRNA is suitable for analysis of gene expression by quantitative reverse tran-scriptase-polymerase chain reaction (RT-PCR) or differential display RT-PCR.

2. MaterialsTo prevent RNase contamination, gloves are worn when handling tissue and

materials. RNase-free glassware and plasticware are used. Solutions are madewith diethyl pyrocarbonate (DEPC)-treated water and autoclaved.

2.1. Equipment for RNA Extraction1. Mortar and pestle.2. Liquid nitrogen.3. Ultracentrifuge (e.g., Beckman).4. Rotor (e.g., SW60Ti).

2.2. Reagents for RNA Extraction1. Guanidinium isothiocyanate (GIT).2. Homogeneization buffer: 4 M GIT, 25 mM sodium citrate, pH 7.0, 0.5% N-lauroyl-

sarcosine, 0.1% -mercaptoethanol.3. Phenol (Rnase-free).4. Chloroform/Isoamyl alcohol (24:1).5. Proteinase K.6. Cesium trifluoroacetate (CsTFA).7. RNase-free DNase (Promega).8. Rnase inhibitor RNasin (PE Applied Biosystems).9. Sodium acetate.

10. DEPC-treated water: dissolve 1 g DEPC in 1 L distilled water, and then auto-clave for 40 min at 128°C. (DEPC inactivates Rnases.)

3. Methods3.1. RNA Extraction

1. Freeze and store cartilage samples (50–150 mg) into liquid nitrogen.2. Reduce the frozen samples to powder with a mortar and pestle previously cooled

in liquid nitrogen.3. Isolate total RNA by using a combination and modification of GIT procedures

(1,2). Homogenize the frozen samples in 5 mL of homogeneization buffer for 3 hat 4°C.

4. Phenol/chloroform extraction: add to the sample 5 mL of phenol and 2.5 mL ofchloroform/isoamyl alcohol. Mix by vortex and centrifuge at 10,000g for 1 h at4°C. Carefully remove the upper phase.

5. Precipitation: add 1/10 vol of 3 M sodium acetate, pH 5.2, and 2 vol of frozen100% ethanol. Mix and incubate at –20°C overnight. Centrifuge at 10,000g for30 min at 4°C.

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6. Resuspend the pellets in 0.8 mL of 1 M GIT with 200 μg/mL proteinase K, andincubate at 40°C until complete dissolution (see Note 1).

7. Adjust GIT concentration to 4 M by adding 1.2 mL of 6 M GIT, layer the sampleson a cushion of CsTFA with a density of 1.6 g/mL, and ultracentrifuge in aBeckman SW60Ti rotor at 88,000g for 18 h at 18°C.

8. To eliminate possible traces of genomic DNA, resuspend RNA pellets in a totalvolume of 200 μL for digestion with 6 U of RNase-free DNase in the presence of80 U of RNasin, for 30 min at 37°C.

9. Finally recover total RNA after a further phenol-chloroform extraction and sodiumacetate precipitation. Resuspend the pellet in 10–15 μL DEPC-treated water andassay 1 μL for concentration and purity of RNA by measuring A260/A280.

4. Notes1. For our preliminary analyses of gene expression by RT-PCR in human or animal

cartilages, we used classical protocols described in the literature for RNA extrac-tion. We obtained extremely low yields of RNA, and our RT-PCR amplification

Fig. 1. Agarose-gel resolution patterns of total RNA isolated from bovine cartilagewithout (A) or with (B) CsTFA centrifugation. (A) Frozen cartilage samples werehomogeneized in a solution containing 4 M GIT. After phenol-chloroform extractionand precipitation, the pellets were resuspended in 1 M GIT with 200 μg/mL proteinaseK and incubated at 40°C until complete dissolution. The samples were again phenol-chloroform extracted and precipitated. RNA preparations were electrophoresed informaldehyde-agarose (1%) minigel and stained with ethidium bromide (lane 1) ortransferred to nylon membranes for Northern blot hybridization with a type II collagenprobe (lane 2). Note that this RNA is suitable for a good-quality hybridization. Afterethidium bromide staining, the sample shown in (lane 1) was further stained with tolui-dine blue. Toluidine blue reveals contamination of proteoglycans (PGs) in an RNApreparation (lane 3). (B) Electrophoresis and toluidine blue staining of RNA isolatedas indicated in Subheading 3.1. Note that PGs have been eliminated by ultracentrifu-gation. The positions of the 28S, 18S, and 5S ribosomal RNAs are shown.

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reactions were poorly reproduced, very likely because of contamination by remain-ing proteoglycans. We found that a treatment by proteinase K in 1 M GIT prior toCsTFA ultracentrifugation was crucial to increase the yield and purity of RNAextracted from human, bovine, mouse, rabbit, or chick cartilage. An example isshown in Fig. 1. In particular, this method led to reproducible patterns of conven-tional PCR after reverse transcription of minute amounts of RNA, which is espe-cially convenient for human samples, which are difficult to obtain in high quantities(3,4). Moreover, this protocol allowed us to obtain reproducible patterns of differ-ential display RT-PCR and should be useful for identifying genes differentiallyexpressed by chondrocytes in situ (5).

References1. Chomczinski, P. and Sacchi, N. (1987) Single-step method of RNA isolation by

acid guanidinium thiocyanate-phenol-chloroform extraction. Anal. Biochem. 162,156–159.

2. Adams, M. E., Huang, D. Q., Yao, L. Y., and Sandell, L. J. (1992) Extraction andisolation of mRNA from adult cartilage. Anal. Biochem. 202, 89–95.

3. Bluteau, G., Labourdette, L., Ronzière, M. C., et al. (1999) Type X collagen inrabbit and human meniscus. Osteoarthritis Cartilage 7, 498–501.

4. Bluteau, G., Conrozier, T., Mathieu, P., Vignon, E., Herbage, D., and Mallein-Gerin, F. (2001) Matrix metalloproteinase-1, -3, -13 and aggrecanase-1 and -2 aredifferentially expressed in experimental osteoarthritis. Biochim. Biophys. Acta1526, 147–158.

5. Bluteau, G., Gouttenoire, J., Conrozier, T., et al. (2002) Differential gene expres-sion analysis in a rabbit model of osteoathritis induced by anterior cruciate liga-ment (ACL) section. Biorheology 39, 247–258.

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