13.2.1 Primer Design
The four biotinylated oligo(dT) anchored primers (T12VN) are designed as follows where V represents mixed bases of A, G, and C:
The arbitrary decamers can be designed by randomly picking ten bases with about 60% GC content. Different arbitrary decamer kits are also available commercially (Operon Technologies, Alameda, CA).
Total RNA is isolated from normal and prostate cancer specimens by the single-step, acid guani-dinium thiocyanate-phenol-chloroform extraction method10 or by TRIZOL reagent (GIBCO/BRL, Gaithersburg, MD) following manufacturer's directions. RNA (10 jig) from each tissue is treated with 5 units of RNase-free DNase I (GIBCO/BRL, Gaithersburg, MD) in the presence of 20 mM Tris-HCl, pH 8.4, 50 mM KCl, 2 mM MgCl2, and 20 units of RNase inhibitor (Boehringer Mannheim, Indianapolis, IN). After extraction with phenol/chloroform and ethanol precipitation, the RNA is redissolved in DEPC-treated H2O. Four pools of cDNA are generated using the four different biotinylated oligo(dT)-anchored primers. For each pool, five jig of the DNase-treated RNA from each tissue is reverse-transcribed into cDNA using one of the biotinylated oligo(dT)-anchored primer and M-MLV reverse transcriptase (GIBCO/BRL) in a total of 40 jl reaction. The reaction mixture contains 50 mM Tris-HCl, pH 8.3, 75 mM KCl, 3 mM MgCl2, 10 mM DTT, 500 jM dNTP, 2 jM biotinylated oligo(dT)-anchored primer, and 400 U M-MLV reverse transcriptase. The reactions are incubated at room temperature (22°C) for 10 min, followed by a 50-min incubation at 42°C. A final 10-min incubation at 70°C is performed to inactivate the enzyme.
For each pool of cDNA from different cells or tissues (e.g., normal and cancer), PCR is performed using the same biotinylated oligo(dT)-anchored primer as in reverse transcription, combining with an arbitrary 10mer. Different primer combinations can be used for different PCR reactions to generate distinct band patterns. The PCR mixture contains 2 jl of cDNA, 10 mM (Tris)-HCl, pH 9.3, 50 mM KCl, 1.5 mM MgCl2, 50 jM dNTPs, 1.25 U of Taq DNA polymerase, and 0.2 jM each of the biotinylated oligo(dT)-anchored primer and arbitrary 10mer in a total of 20 jl reaction. The amplification parameters include 40 cycles of reaction with 30 s denaturing at 94°C, 2 min annealing at 40°C, and 1 min extension at 72°C. A final extension at 72°C is performed for 15 min. After PCR, 12 jl of loading dye (95% formamide, 0.05% bromophenol blue, and 0.05% xylene cyanol) is added to each tube.
13.2.4 Electrophoresis and Chemiluminescent Detection
A 6% denaturing polyacrylamide DNA sequencing gel is prepared following established procedure.1 The gel is prerun for about 1 h to warm up to about 50°C. The PCR products with loading dye are heated at 80°C for 3 min, and 5 jl of each is loaded immediately onto the gel. The gel is run at about 120 W constant power for about 2 h or until the xylene cyanol dye reaches about 20 cm from the bottom.
After electrophoresis, DNA fragments on the gel are transferred to the Tropilon-PlusTM nylon membrane (Tropix), or other positive-charged nylon membrane by capillary transfer. Fixing the gels or removing the urea is unnecessary. Approximately 20% of the DNA is transferred to the membrane, which can be easily detected using chemiluminescent detection procedure. The setup for capillary transfer is illustrated in Figure 13.1 and the procedure is as follows:
1. Disassemble the gel apparatus and separate the glass plates.
2. Lay a piece of Whatman® 3MM filter paper on top of the gel, making sure the paper is in good contact with the gel. Remove the gel from the glass plate with the filter paper. Place the paper with gel attached back on the plate with the gel side up.
3. Cut a piece of nylon membrane the same size as the region of the gel to be blotted and wet it thoroughly with TBE.
4. Carefully place the wet membrane on the gel. Remove any air bubbles by rolling a pipette over the membrane.
5. Place three pieces of dry Whatman filter paper on top of the membrane, another glass plate, and a 2-kg weight on top. Allow the transfer to proceed for 1 h.
Following the transfer step, orient the membrane and the gel by punching through using a needle with India ink in three locations. The membrane is then carefully separated from the gel. The transferred DNA is immobilized on the membrane by UV irradiation (total exposure: 120 mJoules), or baking at 80°C for 1 h.
The SEQ-LightTM chemiluminescent detection system (Applied Biosystems) is used to detect bands on the sequencing gel following manufacturer's instruction. After detection, DNA band patterns are captured on X-ray film, and differential expressed bands are identified.
13.2.5 Reamplification, Sequencing, and Confirmation of Expression
After positive bands are identified, the X-ray film and the nylon membrane are then used to locate the band position back to the gel. The remaining DNA (about 80%) on the gel in the identified position is recovered. Reamplification is performed following established procedure.3,4 Briefly, each gel slice is soaked in 100 fl of H2O in a 1.5-ml centrifuge tube and boiled for 10 min. After centrifuging for 2 min, the supernatant is transferred to a new tube. The DNA is precipitated by adding 10 f l of 3 M NaOAc (pH 5.2), 2 fl of glycogen (20 mg/ml), and 250 fl of ethanol, followed by incubating at dry ice or -80°C freezer for 20 min and then spinning for 10 min. The pellet is washed with 500 fil of 70% ethanol, dried, and resuspended in 10 fl of H2O. Reamplification is performed using 4 fl of the DNA and the same set of primers and PCR conditions as in the display experiment. The reamplified DNA band can be used directly as a probe for Northern confirmation or a template for DNA sequencing.
Since all the antisense DNA molecule in the reamplified product has a biotin in its 5' end, the reamplified DNA can be used directly as probe for non-isotopic Northern hybridization to confirm the differential expression of the gene. The reamplified band is purified by Qiaex kit (Qiagen, Chatsworth, CA) and eluted into 20 fl of H2O. Northern hybridization is performed using established procedure.1 Then, 10 fl of the purified DNA is boiled for 5 min, cooled on ice, and added to hybridization solution. Following hybridization and washing, the signal can be detected by chemiluminescent detection. The purified DNA band can also be used as a probe for non-isotopic cDNA library screening to clone full-length cDNA of the gene.
The purified band can be sequenced directly using the same biotinylated oligo(dT)-anchored primer and SeqLight® system. After DNA sequence data are obtained, primers can be designed and used in a relative reverse transcription-PCR (RT-PCR) experiment to confirm the differential expression of the gene, or used in a Rapid Amplification of cDNA End (RACE) experiment to clone the full-length cDNA of the gene.
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