Differential Display of Cotton Transcripts

Song, P., Yamamoto, E., and Allen, R. (1995) Plant Mol. Biol. Rep. 13:174-181

Plant Materials

• RNase-free DNase I (1000 units/ml, Promega, Madison, WI)
• RNasin (recombinant ribonuclease inibitor, 2600 units/ml, Promega, Madison, WI)
• DNase I buffer: 100 mM Tris-HCl (pH 8.0), 25 mM NaCl, 15 mM MgCl₂, 0.25 mM CaCl₂, 2.5 mM KCl
• PCI: phenol:chloroform:isoamyl alcohol (25:24:1)
• CI: chloroform:isoamyl alcohol (24:1)
• 7.5 M ammonium acetate
• Moloney murine leukemia virus reverse transrciptase M-MuLV RT (20000 units/ml, Promega, Madison, WI)
• 5X M-MuLV RT buffer (Promega, Madison, WI)
• 20 uM decamer primers (random)
• 20 uM anchored oligo-dT12MN primers (M=A,C, or G; N=A,C,G, or T)
• mixed dNTPs (2.5 mM each dNTP, 100 uM each dNTP
• Taq DNA Polymerase and buffer (Promega, Madison, WI)
• 25 mM MgCl₂
• alpha-[33P]-dATP (NEN Research Products, du Pont de Nemours & Co, Boston, MA)
• 40% acrylamide:bisacrylamide (29:1) stock solution (Fisher Scientific, Houston, TX)
• TBE buffer: 0.09 M Tris-borate, 0.002 M EDTA (pH 8.0)
• sample loading solution: 0.25% bromophenol blue, 0.25% xylene cyanol FF, 40% (w/v) sucrose in water
• TA cloning kit (pCRII vector system, Invitrogen, San Diego, CA, or pGEM-T vector system, Promega, Madison, WI)
• TAE buffer: 0.04 M Tris-acetate, 0.001 M EDTA (pH 8.0)

DNase I Treatment

• To a 1.5 ml microcentrifuge tube, add 30 to 50 ug of RNA, 40 ul of DNase I reaction buffer, 5 ul RNasin, 10 ul DNase I, bring to total volume of 100 ul with DEPC treated water, mix gently.
• Incubate 30 min in a 37C water bath.
• Purify the RNA by extraction with PCI and CI.
• Add 1/2 volume of 7.5 M ammonium acetate, mix well.
• Add 2 volume of cold ethanol, precipitate the RNA at -20C for at least 1 hour.
• Centrifuge at 10000X g for 15 min at 4C, discard ethanol, air dry the pellet, and resuspend in 40 ul DEPC-treated water.

First-strand DNA Synthesis

• To a 0.5 ml microcentrifuge tube, add 2 ug of DNase I-treated total RNA, bring to 25 ul with DEPC treated water.
• Heat the tube in 65C water bath for 10 min.
• Add 10 ul 5X M-MuLV RT buffer, 5 ul 2.5 mM mixed dNTPs, 6 ul 20 uM anchored oligo-dt primer, 1 ul RNasin.
• Incubate at room temperature for 15 min.
• Add 1.5 ul M-MuLV RT, gently mix.
• Incubate at 37C for 1 hour.
• Heat the reaction in 95C water bath for 5 min, store cDNA at -20C for later use.

PCR Amplification

• To a 0.5 ml microcentrifuge tube, add 4.0 ul cDNA, 2.5 ul 100 uM mixed dNTPs, 2.5 ul 10X Taq DNA polmerase buffer, 2.5 ul 25 mM MgCl₂, 2.5 ul 20 uM anchored oligo-dT primer (same as was used in the cDNA synthesis), 1.0 ul 20 uM 10-mer arbitrary primer, 0.5 ul alpha-[33P]dATP (10 mCi/ml), 0.5 ul Taq DNA polymerase, bring the reaction to 25 ul with sterile water, gently mix, cover the reaction with 25 ul sterile mineral oil (if needed).
• Run PCR reaction as follows: 94C for 30 sec, 42C for 1 min, 72C for 30 sec, 40 cycles, followed by a 5 min final extension at 72C.

Non-denaturing Sequencing Gel Electrophoresis

• Pour a 6% non-denaturing polyacrylamide sequencing gel (same as regular sequencing gel but without urea).
• Pre-run the gel in 1X TBE buffer at 30 mA for 30 min.
• Add 8 ul sample loading solution to each reaction, mix well, heat at 72C water bath for 2-3 minutes prior to loading.
• Run the gel at 30 mA until xylene cyanol reaches the bottom of the gel.
• Dry the gel without fixing, tape the dried gel and X-ray film together, mark the film and the gel by punching several holes with a needle.
• Expose the film for 8-10 hours (or overnight) at -80C with intensifying screens.

Recovery, Reamplification, and Cloning of cDNA fragments

• Align the developed film with the gel, cut differentially displayed bands from the dried gel with a razor blade, cut into small pieces, and put them into a 1.5 ml microcentrifuge tube.
• Add 200 ul sterile water (good for two 1.5mm x 7mm slices), incubate for 15 min at room temperature.
• Boil the tube for 10 min.
• Centrifuge 1 min at 10000X g, transfer the supernatant to a fresh tube, and pipette 10 ul into another fresh microcentrifuge tube.
• Reamplify this 10 ul of eluted DNA in a 25 ul reaction volume using the same primers and PCR conditions as used above, but without radiolabelled dATP.
• Remove 10 ul of reamplified PCR product, run on a 1% agarose gel in 1X TAE buffer, stain the gel with ethidium bromide (10 mg/ml).
• Ligate the remaining PCR products into a TA cloning vector (pCRII or pGEM-T), and transform competent Escherichia coli (e.g. DH5) cells.