cDNA/AFLP：A tool for transcriptome analysis

RNA Isolation

1.1.Plant tissue material is harvested and immediately frozen in liquid N ₂ . The material is then ground to a fine powder in a pre-cooled pestle and mortar under liquid N ₂ . (This material can be stored at -80°C indefinitely). To proceed with extraction, transfer around 1g of powdered material into pre-cooled 50 ml plastic tubes (usually around 5 ml volume).

1.2.Transfer 10 ml of a hot (80°C) 1:1 mixture of RNA extraction buffer and phenol (base volume: 5 ml) into the tube containing plant material . Be sure that no liquid N ₂ remains in the sample when adding the extraction buffer . Vortex or manually shake the samples vigorously for at least 30 seconds.

1.3.To the mixture add a base volume of chloroform (5 ml) and shake vigorously. To separate the phases, centrifuge the samples for 20 minutes (3500 rpm in a large swing-out rotor). Repeat chloroform extraction with one base volume (5ml) until no inter-phase can be detected (at least twice).

1.4.Transfer resultant cleared aqueous phase to a new 50 ml tube (ca. 5.5 ml; this material can be stored frozen for 24 h). Add 1/3 volume of ice cold 8M LiCl and precipitate for at least 3 h at 0°C (or over night). Centrifuge RNA at 0°C for 20 minutes (3500 rpm) and resuspend in 500 µl H ₂ O after extensive washing of the pellet with 75% Ethanol (ice cold). Take a small aliquot of this sample or determine concentration, integrity and purity of the RNA (1µl for agarose gel and 1µl for spectrophotometer).

1.5.Precipitate RNA in 2 volumes 96% Ethanol in the presence of 1/10 volume 3 M Na Acetate pH 5.3 for 1h and centrifuge. Resuspend the RNA pellet thoroughly in H ₂ O (heat for 5 minutes at 65°C to help resuspension if necessary) to obtain a 1 µg/µl RNA solution

2. Bead preparation (poly d[T] ₂₅ V beads) (Dynabeads M-280 streptavidin, Dynal Biotech)

2.1. Use 0.5 mg (50 µl) of bead suspension per 50 µg total-RNA. Wash the beads twice in an equal volume of STEX buffer. Resuspend the beads in 2 vol. of 2x-STEX buffer (100 µl).
2.2.Add 2 vol (100 µl) of biotinylated d[T]25V oligonucleotide (Oligo00) [10ng/µl] (20 ng oligo00/µl beads). 1 mg of streptavidine beads binds 200 pM of biotinylated d[T] ₂₅ V oligonucleotide (Oligo00) thus, 2000 ng of Oligo00 gives 1.5 excess. Incubate at room temperature for 30’.
2.3.Wash the beads three times with 2 vol of STEX buffer (100 µl) to eliminate unbound Oligo0. After the last wash, resuspend beads in 1 vol (50 µl) of 2x-STEX buffer.

3. ¹ Poly-A ⁺ RNA enrichment and cDNA synthesis
3.3. Take 50 μg of total RNA [1 μg/μl] and add to the 50 μl beads prepared previously. Incubate at room temperature for 10’and subsequently on ice for 5’.
3.4. Wash bead / RNA mixture 3 times in STEX buffer (100 μl). Eliminate all traces of STEX buffer by centrifugation after the last wash. Finally, resuspend the beads in 20 μl of H ₂ O.
3.5. Incubate beads at 65°C for 5 min and immediately place in MPC and transfer the 20μl poly-A ⁺ solution into a new tube. (Usually < 20 μl). (Alternatively, two elutions with 10 μl H ₂ O at 65ºC can be done instead).
( ¹ Poly-A ⁺ RNA isolation is not required for the procedure, especially when isolating RNA from small amounts of tissue. However, when total RNA is used for template preparation, DNaseI treatment is required.)

3.6. Prepare the following first strand cDNA synthesis reaction mix:
20.00 μl Poly A+ RNA
1.00 μl primer d[T] ₂₅ V (from 100 ng/μl stock)
6.00 μl 5 x cDNAI buffer (Superscript II buffer)
1.00 μl dNTPs (25 mM)
1.00 μl Reverse transcriptase (200 U, Invitrogen SuperScript II)
2.00 μl DTT (100 mM)
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31.00 μl Incubate for 1 h at 42℃