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Serial Analysis of Gene Expression 273 Table 1 Useful Web Sites for SAGE URL Contents http://www.sagenet.org/sage_protocol.htm Johns Hopkins SAGE protocol and software for non-commercial use, plus conferences, publications and other information (1,2) http://www.genzyme.com/sage/ Genzyme Molecular Oncology: information on SAGE and its commercial use http://www.invitrogen.com Invitrogen including I-SAGE protocol http://www.dsv.cea.fr/thema/get/ SADE: a SAGE Adaptation for Downsized sade.html Extracts (6) http://www.ambion.com Ambion website with advice on RNA work http://www.ncbi.nlm.nih.gov/SAGE/ NCBI’s SAGEmap (20) http://www.ncbi.nlm.nih.gov/UniGene/ NCBI’s UniGene (“unique gene”: clusters all index.html transcripts of one gene under one name) (19) http://www.geneontology.org Gene Ontology databases: information on gene function and cellular location (25) comparisons and for investigation of the expression of individual transcripts, which can be further studied via Unigene (see Table 1, ref. 19). The statistical basis for designing and analyzing SAGE experiments has also been discussed in detail (21–24). Here, however, the focus is on the wet laboratory work. 2. Materials The reagents specified have been used successfully, but many equivalents are available and could be used if preferred (see Note 1). This description is based on the Johns Hopkins protocol (version 1.0d); the most helpful modifications are also described. 2.1. RNA Work (see Note 2) 1. Aerosol-resistant pipet tips (Greiner Labortechnik Ltd, Gloucestershire, UK). 2. DEPC-treated water (Diethylpyrocarbonate: Sigma, Dorset, UK). 3. (Optional) RnaseZap ® (Ambion (Europe) Ltd, Cambridgeshire, UK). 2.2. Kits for Purification of RNA and cDNA Synthesis 1. Preparation of total RNA: TRIZOL ® Reagent (Invitrogen Life Technologies, Paisley, UK). 2. Purification of polyA+ mRNA: Poly(A)Purist Kit (Ambion). 3. (Alternative protocols: streptavidin-coated tubes obtained with mRNA Capture Kit or separately (Roche Diagnostics Ltd, East Sussex, UK), instead of steps 1 and 2 (see Note 3 and Subheadings 2.4. and 2.6.). 4. cDNA synthesis: SUPERSCRIPT Choice System for cDNA Synthesis (Invitrogen). 2.3. Purification of DNA 1. Phenolchloroform (P/C): phenolchloroformisoamyl alcohol (25241; Ambion). 2. Glycogen, molecular biology grade (20 mg/mL) (Roche Diagnostics Ltd). 3. Ammonium acetate (10 M).
274 Oien 4. 100% and 70% ethanol. 5. LoTE: 3 mM Tris-HCl (pH 7.5), 0.2 mM EDTA (pH 7.5). 6. TE: 10 mM Tris-HCl (pH 7.5), 1 mM EDTA (pH 7.5). 7. (Optional) QIAquick ® DNA Cleanup System: QIAquick Nucleotide Removal and PCR Purification Kits; and, for higher through-put, QIAquick 8 PCR Purification Kit with QIAvac 6S (Qiagen Ltd, West Sussex, UK). 2.4. Oligonucleotides 1. Biotinylated oligo dT: 5′ [biotin] T 18 (Alternative protocols, see Note 3). 2. SAGE Linker 1A: 5′ TTT GGA TTT GCT GGT GCA GTA CAA CTA GGC TTA ATA GGG ACA TG 3′ 3. SAGE Linker 1B: 5′ TCC CTA TTA AGC CTA GTT GTA CTG CAC CAG CAA ATC C [amino mod. C7] 3′ 4. SAGE Linker 2A: 5′ TTT CTG CTC GAA TTC AAG CTT CTA ACG ATG TAC GGG GAC ATG 3′ 5. SAGE Linker 2B: 5′ TCC CCG TAC ATC GTT AGA AGC TTG AAT TCG AGC AG [amino mod. C7] 3′ 6. SAGE Primer 1: 5′ [biotin] GGA TTT GCT GGT GCA GTA CA 3′ (11) 7. SAGE Primer 2: 5′ [biotin] CTG CTC GAA TTC AAG CTT CT 3′ (11) 8. M13 Forward: 5′ GTA AAA CGA CGG CCA GT 3′ 9. M13 Reverse: 5′ GGA AAC AGC TAT GAC CAT G 3′ The working concentration of all primers, except biotinylated oligo dT, is 350 ng/µL. Obtain linkers and biotinylated oligos gel-purified: recommended suppliers include Integrated DNA Technologies, Inc. (IA, USA) and Oswel Research Products Ltd. (Southampton, UK). Before use, linkers must be kinased, either biochemically during synthesis or later enzymatically (see Note 4), and annealed to the complementary linker. 2.5. Restriction and Modifying Enzymes 1. NlaIII (10 U/µL), supplied with NEBuffer 4 and 100× bovine serum albumin (New England Biolabs [NEB] Inc, MA,) stored at –70°C). 2. BsmFI (2 U/µL) (NEB). 3. SphI (5 U/µL) (NEB). 4. T4 Polynucleotide Kinase 10U/µL (NEB) (or obtain linker oligonucleotides already kinased, see Note 4). 5. T4 DNA Ligase (5 U/µL, or 20 U/µL if preferred; NEB). Aliquot ligase enzyme and buffer to prevent cross-contamination from PCR products. 6. DNA polymerase I large fragment (Klenow); 5 U/µL (NEB). 2.6. Magnetic Beads and Related Materials 1. Dynabeads ® M-280 Streptavidin (-coated) (Dynal AS, Oslo, Norway). 2. Magnetic particle concentrator (magnetic stand to immobilize beads) and sample mixer (Dynal AS). 3. 2× Binding and washing (B+W) buffer: 2 M NaCl, 10 mM Tris-HCl (pH 7.5), 1 mM EDTA. Also prepare 1× B+W buffer solution. 4. (Optional) Nonstick RNase-free tubes (Ambion) have been recommended for use with magnetic beads to minimize adhesion of beads to the tube walls. 5. (Alternative protocols: Dynabeads ® Oligo dT 25 (Dynal AS; see Note 3 and Subheadings 2.2. and 2.4.).
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TM Methods in Molecular Biology Vol
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Xin Wang and W. Scott Young III 105
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63. Primed In Situ Nucleic Acid Lab
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4 Bartlett and Stirling The thing t
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6 Bartlett and Stirling Fig. 2. Res
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8 Carroll and Casimir of PCR. Such
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10 Carroll and Casimir cost more th
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12 Carroll and Casimir are no longe
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14 Carroll and Casimir Should these
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16 McDonagh Fig. 1. Unidirectional
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18 McDonagh stored. This means that
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20 McDonagh
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22 Stirling which will either not a
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24 Stirling
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28 Bartlett References 1. US Depart
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30 Bartlett and White 11. 5 M sodiu
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32 Bartlett and White
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34 Pearson and Stirling 11. Microfu
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36 Going 3. Methods 3.1. Section Cu
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38 Going pipet filler. Spread the p
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40 Going digitally to record the di
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42 Going
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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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48 Pearson 3. Method The method of
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50 Stirling and Bartlett 4. Protein
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52 Stirling and Bartlett
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54 Stirling 3. Methods 3.1. Fungal
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56 McDonagh 2. Add 0.4 mL of TNE bu
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58 Schmerer 2. Materials To apply t
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60 Schmerer 3. The additional purif
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62 Schmerer
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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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72 Bartlett 7. Remove upper aqueous
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74 Bartlett 4. Many modern electrop
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76 Bartlett
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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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84 Hyndman and Mitsuhashi Fig. 1. H
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86 Hyndman and Mitsuhashi Fig. 3. H
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88 Hyndman and Mitsuhashi can be ge
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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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96 Grunenwald than absolutely neces
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98 Grunenwald 2. Foord, O. S. and R
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100 Grunenwald
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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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110 Wang and Young Fig. 3. Expresse
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112 Wang and Young 2. First-strand
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114 Wang and Young 3′-RACE outlin
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116 Wang and Young
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118 Dassanayake and Samaranayake co
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120 Dassanayake and Samaranayake 5.
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122 Dassanayake and Samaranayake Re
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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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134 Iannone et al.
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136 Benjamin, Smith, and Waites amp
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138 Benjamin, Smith, and Waites the
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140 Benjamin, Smith, and Waites 2.2
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142 Benjamin, Smith, and Waites 2.
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148 Benjamin, Smith, and Waites 8.
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150 Benjamin, Smith, and Waites
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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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164 Abe Table 2 Detection Rate of H
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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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170 Tellier et al. 2. Cline, J., Br
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172 Tellier et al. 38. Tamiya, S.,
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174 Tellier et al. 13. Thin-wall PC
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176 Tellier et al. 13 min for the l
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178 Tellier et al.
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182 Stirling efficiency of the reac
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184 Stirling
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186 Cremer and Moos Fig. 1. Rearran
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188 Cremer and Moos 4. Count cells
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190 Cremer and Moos Fig. 3. Alignme
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192 Cremer and Moos Fig. 4. Testing
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194 Cremer and Moos 5. Compare the
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196 Cremer and Moos 11. Klein, E.,
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198 McDonagh the dilution method is
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200 McDonagh 2.2. Basic PCR 1. dNTP
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202 McDonagh Fig. 2. Quantitative P
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204 McDonagh
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206 Bartlett Table 1 Example Assay
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208 Bartlett 3.4. Calculation of Re
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210 Bartlett 11. Cerenkov counting
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212 Kerr Fig. 1. Fluorescent probes
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214 Kerr after the PCR. This reduce
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218 Bartlett As with any experiment
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220 Bartlett Even once novel regula
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Page 221 and 222: 226 Dominguez et al. Table 1 Primer
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Page 231 and 232: 236 Dominguez et al. 11. Joshi, C.
Page 233 and 234: 238 Khalturin, Kuznetsov, and Bosch
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Page 243 and 244: 248 Ringquist et al. 5. Total RNA s
Page 245 and 246: 250 Ringquist et al. 3. Purificatio
Page 247 and 248: 252 Ringquist et al. 2. The present
Page 249 and 250: 254 Ringquist et al.
Page 251 and 252: 256 Case-Green, Pritchard, and Sout
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Page 281 and 282: 288 Edwards and Bartlett technology
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Page 289 and 290: 296 Rithidech and Dunn 11. Ethidium
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Page 303 and 304: 310 Schmerer the investigation conc
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Page 309 and 310: 316 Aubele and Smida 2.2. Chemicals
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Page 313 and 314: 320 Nakashima, Akahoshi, and Tanaka
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326 Stirling
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328 Han and Robinson Fig. 1. Basic
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330 Han and Robinson sequence diffe
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332 Han and Robinson 4. Notes 1. PC
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334 Han and Robinson
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338 Stirling of simple and improved
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340 Stirling concentrations interfe
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342 Daniels to sequence a 500-bp PC
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344 Daniels 4. Load all of the samp
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346 Daniels References 1. Orita, M.
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348 Kösel et al. Fig. 1. Schematic
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350 Kösel et al. 3. Methods 3.1. P
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352 Kösel et al. Fig. 2. Sequencin
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354 Kösel et al. 5. Kwok, S. and H
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356 Mazars and Theillet Fig. 1. Sch
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358 Mazars and Theillet of genomic
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360 Mazars and Theillet
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362 Suomalainen and Syvänen Fig. 1
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364 Suomalainen and Syvänen detect
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366 Suomalainen and Syvänen temper
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368 Shen et al. ladder. Also, direc
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370 Shen et al. 3. Mineral oil. 4.
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372 Shen et al. extended primers, w
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374 Quivy and Becker Fig. 1. The us
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376 Quivy and Becker that decreases
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378 Quivy and Becker 2. Solution of
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380 Quivy and Becker 7. Precipitate
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382 Quivy and Becker 7. Prepare a p
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384 Quivy and Becker
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386 Walsh by most, but not all M13
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388 Walsh PCR products are now flus
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390 Walsh 5. For palindromic restri
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392 Walsh
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394 McAleer, Coffey, and Dunham Fig
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396 McAleer, Coffey, and Dunham Tab
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398 McAleer, Coffey, and Dunham Tab
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400 McAleer, Coffey, and Dunham
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402 Stirling 5. Homopolymer Regions
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406 Speel, Ramaekers, and Hopman Ta
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408 Speel, Ramaekers, and Hopman Fi
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410 Speel, Ramaekers, and Hopman po
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Table 2 Comparison of PRINS, in Sit
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414 Speel, Ramaekers, and Hopman te
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Table 3 Summary of Control Experime
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418 Speel, Ramaekers, and Hopman 3.
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420 Speel, Ramaekers, and Hopman ad
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422 Speel, Ramaekers, and Hopman 25
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426 Bull and Paskins Fig. 1. Result
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428 Bull and Paskins 2.3. Detection
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430 Bull and Paskins 6. Shake the s
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432 Bull and Paskins
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434 Wiedorn and Goldmann Fig. 1. IS
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436 Wiedorn and Goldmann 41. Protei
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438 Wiedorn and Goldmann 2. Incubat
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440 Wiedorn and Goldmann 3.15. Prim
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442 Wiedorn and Goldmann ficient se
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444 Wiedorn and Goldmann 25. Kommin
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446 Gilchrist and Befus Fig. 1. Sch
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448 Gilchrist and Befus 2. Cells ar
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450 Gilchrist and Befus Fig. 3. RT
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452 Gilchrist and Befus References
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454 Speel, Ramaekers, and Hopman of
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456 Speel, Ramaekers, and Hopman Fi
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462 Speel, Ramaekers, and Hopman bu
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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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476 Horton, Raju, and Conti-Fine Fi
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478 Horton, Raju, and Conti-Fine th
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480 Horton, Raju, and Conti-Fine 1.
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482 Horton, Raju, and Conti-Fine ot
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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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490 Preston Fig. 1. Design of degen
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492 Preston 2. Remove the AmpliTaq
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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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498 Preston 21. Hung, T., Mak, K.,
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500 Ravassard et al. Fig. 1. Constr
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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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522 Jones and Winistorfer The yield
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524 Jones and Winistorfer 7. Goulde
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526 Burke and Barik Fig. 1. The bas
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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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