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AU-Differential Display 225 36 AU-Differential Display, Reproducibility of a Differential mRNA Display Targeted to AU Motifs Orlando Dominguez, Lidia Sabater, Yaqoub Ashhab, Eva Belloso, and Ricardo Pujol-Borrell 1. Introduction AU-rich elements (AREs) are found in 3′ untranslated regions (3′ UTR) of many highly unstable mRNAs for mammalian early-response genes. The minimal AU sequence core within the ARE is the heptamer WAUUUAW, although from a functional point of view, several pentanucleotides clustered in close proximity are the key sequence motif that mediates mRNA degradation (1). Genes containing AREs are of potential biological and pharmacological interest because they often code for inflammatory mediators, cytokines, proto-oncoproteins, and transcription factors (2–5). A targeted differential display named AU-motif directed display (AU-DD) is described here. It allows the isolation of cDNA fragments from ARE-containing mRNAs with minimal sequence information. AU-DD combines a high specificity gained at the polymerase chain reaction (PCR) level with the advantages of direct comparison between samples at different stages of activation to detect differentially expressed genes (6,7). Previous examples of targeted differential display have used longer and wellconserved motifs from multigene families. These include zinc finger motifs (8,9), plant MADS boxes (10), and motifs specific for heat shock proteins (11). They employ longer conserved sequences (from 6 to 8 codons) to design primers that can predictably work well on PCR. The PCR step of AU-DD uses primers which contain two distinctive domains, a double AU motif at the 3′ end (AU2 in short) and a guanosine rich 5′ (Table 1). This primer core sequence is previously incorporated as a 5′ tag in the retrotranscription primer (see below). The 5′ domain configures a sticky anchor that increases Tm and stabilizes primer annealing whereas the 3′ confers motif specificity. Both 5′ and 3′ domains contribute in a cooperative way to primer annealing. When the natural primerbinding site was identified on cloned products, it was always found to be a true AU-rich site. The use of the AU2 domain-based primers does not restrict; however, the spectrum of amplified mRNAs to clustered AU-pentamer containing genes. In fact, cDNAs From: Methods in Molecular Biology, Vol. 226: PCR Protocols, Second Edition Edited by: J. M. S. Bartlett and D. Stirling © Humana Press Inc., Totowa, NJ 225
226 Dominguez et al. Table 1 Primer Sequences for Both AU-DD and Control Genes Protocol Primer Designation Sequence (A) Retrotranscription (RT) and PCR Primers Used in AU-DD 1 G7AU2dT (RT) GGGGGGGTATTTATTTA(ACGT)TTTTTTTTT TTTTTT(ACG) 2 G7AU2 GGGGGGGTATTTATTTA G7AU2A GGGGGGGTATTTATTTAA G7AU2C GGGGGGGTATTTATTTAC G7AU2G GGGGGGGTATTTATTTAG G7AU2T GGGGGGGTATTTATTTAT 3 GTGAU2dT (RT) GGTGGGTGGTATTTATTTA(ACGT)TTTTTTT TTTTTTTT(ACG) 4 GTGAU2 GGTGGGTGGTATTTATTTA GTGAU2A GGTGGGTGGTATTTATTTAA GTGAU2C GGTGGGTGGTATTTATTTAC GTGAU2G GGTGGGTGGTATTTATTTAG GTGAU2T GGTGGGTGGTATTTATTTAT (B) Primers Used in Control PCR Experiments IFNα (J00207) IFNα513s GGCCTTGACCTTTGCTTTA IFNα915as CTTCATCAGGGGAGTCTCTGT GAPDH (M33197) GAPDH19s TCTTCTTTTGCGTCGCCAG GAPDH390as AGCCCCAGCCTTCTCCA (A) 1 and 3 are alternative retrotranscription (RT) primers; 2 and 4 list two series of PCR primers compatible with RT primers 1 and 3, respectively. (B) Specific primers for IFNA and GAPD, respectively. carrying single AU motifs were often amplified, and only 15% of cloned cDNAs contained a complete double AU element (12). On its part, the reverse transcriptase primer incorporates a significant feature by including the PCR primer as a 5′ tag. This allows the use of a single primer in the PCR, providing the possibility of a fine titration of the annealing temperature to avoid primer artifacts generated when two oligonucleotides are used in low stringency conditions. The cDNA is synthesized from total RNA trying to ensure initiation from true poly-A tails of mRNA, which is not always easy. With our protocol, only 8% of cloned products lacked the poly-A tail (12). For two mRNA species to be effectively sampled by DD, the following two conditions are critical: (1) comparable amounts of template must be used and (2) cDNA concentration must be above a threshold to obtain reproducible results. During all the initial processes, RNA concentration and integrity and the absence of contaminating DNA has to be monitored carefully. Even trace amounts of genomic DNA can significantly alter the final profile, yielding fake products and turning the technique unreliable. RNA should be checked for quantity and integrity both after purification and after DNase treatment. The use of good-quality DNase does not guarantee the removal of all DNA, and this process requires also monitoring. The assessment of
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28 Bartlett References 1. US Depart
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44 Pearson 7. Add 60 µL of chlorof
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46 Bartlett 3. Methods 3.1. RNA Ext
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56 McDonagh 2. Add 0.4 mL of TNE bu
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60 Schmerer 3. The additional purif
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64 Stirling
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66 Bartlett Table 1 Recommended Aga
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68 Bartlett Table 2 Separation of D
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70 Bartlett 2. 5× TBE: 54 g of Tri
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78 Stirling 11. Store at -20°C for
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82 Hyndman and Mitsuhashi 3.1. Effi
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90 Grunenwald may be affected by ea
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92 Grunenwald 50°C will generally
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94 Grunenwald of template DNA, a su
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102 Stirling Table 1 Thermal Cycler
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104 Stirling
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106 Wang and Young full-length cDNA
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108 Wang and Young Fig. 1. (A) Nort
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118 Dassanayake and Samaranayake co
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120 Dassanayake and Samaranayake 5.
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124 Iannone et al. Fig. 1. Diagram
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Table 1 Oligonucleotides Descriptio
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128 Iannone et al. 5. For microsphe
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130 Iannone et al. 4. To adjust for
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132 Iannone et al. 6. Target concen
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136 Benjamin, Smith, and Waites amp
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152 Olmos et al. Fig. 1. RT-hemines
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154 Olmos et al. This nested RT-PCR
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156 Olmos et al. Table 2 Volume and
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158 Olmos et al. 8. The sensitivity
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160 Olmos et al.
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162 Abe 5. Moloney murine leukemia
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166 Abe 4. For HBV, nested PCR usin
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168 Tellier et al. of Pfu (and ther
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276 Oien 2. Purify polyA + mRNA fro
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278 Oien 50-mL conical tubes. Resus
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280 Oien Forward Primer, and then r
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282 Oien 8. PCR. With SAGE, achievi
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288 Edwards and Bartlett technology
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290 Edwards and Bartlett ing less p
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292 Edwards and Bartlett Stepwise m
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294 Edwards and Bartlett
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296 Rithidech and Dunn 11. Ethidium
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298 Rithidech and Dunn Fig. 1. PCR
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302 Edwards and Bartlett Studies th
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308 Edwards and Bartlett 3. Sartor,
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310 Schmerer the investigation conc
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312 Schmerer References 1. Kunkel,
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320 Nakashima, Akahoshi, and Tanaka
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340 Stirling concentrations interfe
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342 Daniels to sequence a 500-bp PC
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386 Walsh by most, but not all M13
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392 Walsh
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394 McAleer, Coffey, and Dunham Fig
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452 Gilchrist and Befus References
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468 Pearson and Stirling into PCR p
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470 Wang 2. Materials 2.1. PCR 1. D
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472 Wang Fig. 1. A brief outline of
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474 Wang
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484 Horton, Raju, and Conti-Fine
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486 Preston amplification approach
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488 Preston 1. 10× PCR buffer: 100
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494 Preston 3.3. Cloning and DNA Se
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496 Preston MgCl 2 concentrations b
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502 Ravassard et al. size dispersio
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504 Ravassard et al. 9. ddH 2 O. 10
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506 Ravassard et al. 3.2.2. RNA Mix
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508 Ravassard et al. 4. Wash twice
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510 Ravassard et al.
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512 Pont-Kingdon Fig. 1. Constructi
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514 Pont-Kingdon 9. Invert tube and
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516 Pont-Kingdon
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518 Jones and Winistorfer Fig. 1. D
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520 Jones and Winistorfer Fig. 2. D
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528 Burke and Barik concentration o
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530 Burke and Barik purified mutant
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532 Burke and Barik
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