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Direct and Indirect In Situ PCR 435 4. Cover Slips (Omnilab, Germany). 5. Microtome (Leica, Germany). 6. Heating Oven for 220°C (WTB Binder, Germany). 7. Water bath (e.g., GFL 1083, GFL, Germany). 8. Pattex Supermatic 200plus (Henkel, Düsseldorf, Germany). 9. Forceps. 10. Laboratory gas burner. 11. DEPC (Sigma, Deisenhofen, Germany, Cat-No. D5758). 12. 5 NNaOH (Merck, Germany). 13. 1 NHCl (Merck, Germany). 14. NaCl (Merck, Germany). 15. NaH 2 PO 4 H 2 O (Merck, Germany). 16. Na 2 HPO 4 2H 2 O (Merck, Germany). 17. Tris-HCl (Sigma, Deisenhofen, Germany). 18. EDTA (Sigma, Deisenhofen, Germany). 19. 10× phosphate-buffered saline (PBS): 1.3 M NaCl, 5 mM NaH 2 PO 4 , 95 mM Na 2 HPO 4 . Adjust to pH 7.2 with orthophosphoric acid. 20. 5% buffered formalin pH 7.2 (Frontell, Germany). 21. 4% buffered paraformaldehyde, pH 7.2. Dissolve 40 g of paraformaldehyde (Merck ultra pure 4005, Germany) in 1× PBS. Adjust to pH 7.2 with NaOH and fill up to 1000 mL with 1× PBS. Filter through Wathman No 1. 22. 100% ETOH (Merck, Germany). 23. Xylene (Merck, Germany). 24. Taq Polymerase 5 U/µL (Roche Diagnostic GmbH, Mannheim, Germany, Cat No. 1146173). 25. 0.1 M Tris-HCl, 0.1 M NaCl, pH 7.5. 26. 1 M MgCl 2 (Sigma, Deisenhofen, Germany). 27. CaCl 2 (C3306, Sigma, Deisenhofen, Germany). 28. 20× SSC: 3 M NaCl and 0.3 M Na 3 Citrate. Adjust to pH 7.0 with HCl. 29. Bovine serum albumin (BSA) 20 mg/ mL (Roche Diagnostic GmbH, Mannheim, Germany, Cat-No. 711454). 30. 10% SDS (Sigma, Deisenhofen, Germany, Cat-No. L 4522). 31. 100% deionized formamide (Sigma, Deisenhofen, Germany, Cat-No. F9037). 32. Dextran sulfate (Sigma, Deisenhofen, Germany, Cat-No. D8906): 50% dextran sulfate. Dissolve 50 mg in 100 mL of DEPC-treated distilled water. Heat to 60°C and shake to speed up the dissolving. 33. DIG Wash and Block Buffer Set (Roche Diagnostic GmbH, Mannheim, Germany, Cat-No. 1585762) containing 10× washing buffer, 10× blocking solution (=10% blocking solution), 10× detection buffer, 10× maleic acid buffer. 34. NBT 100 mg/mL (Nitroblue tetrazolium chloride; Roche Diagnostic GmbH, Mannheim, Germany Cat-No. 1383213). 35. BCIP 50 mg/mL (5-Brom-4-chlor-3-indoyl-phosphate) (Roche Diagnostic GmbH, Mannheim, Germany, Cat-No. 138221). 36. Mounting media (as supplied; Permount SP15-500, Fisher Scientific, Wiesbaden, Germany). 37. Counterstain (Nuclear Fast Red and Eosin, Merck, Germany). 38. Anti-Dig or Anti-biotin antibody conjugates (Roche Diagnostic GmbH, Mannheim, Germany Cat-No. 1426303, 1426311, 1093274, 1207733). 39. Hybridization probes (Eurogentec, Belgium or MWG <strong>Bio</strong>tech, Ebersberg, Germany). 40. DNAse (Roche Diagnostic GmbH, Mannheim, Germany, Cat-No. 776785).
436 Wiedorn and Goldmann 41. Proteinase K solution: Proteinase K (Roche Diagnostic GmbH, Mannheim, Germany; 250 µg/mL), 100 mM Tris-HCl and 50 mM EDTA. 42. 10× Target Retrieval (Dako, Hamburg, Germany, Cat-No. S1700). 43. DNAse solution: 40 mM Tris-HCl, pH 7.4, 6 mM MgCl 2 , 2 mM CaCl 2 , and 1 U/µL RNAse free DNAse (Roche Diagnostic GmbH, Mannheim, Germany). 44. IL6 PCR mix: 10 mM Tris-HCl, pH 9.2, 50 mM KCl, 3 mM MgCl 2 , 0.2 mM dNTPs (Roche Diagnostic GmbH, Mannheim, Germany); 0.1% BSA (Roche Diagnostic GmbH, Mannheim, Germany); 10 µM DIG-11-dUTP (Roche Diagnostic GmbH, Mannheim, Germany) (see Note 3); 5 U/sample Taq-Polymerase (Roche Diagnostic GmbH, Mannheim, Germany); and 0.4 µM of sense and antisense primer. 45. Sequence of IL6 primers (see Note 4): sense primer: 5′ CTTCTCCACAAGCGCCTTC-3′; antisense primer: 3′ CTAAGTTACTCCTCTGAACGG-5′. 46. A typical hybridization mix is composed of (exact compositions depends on the probe in use): 2× up to 5× SSC; up to 50% formamide; 5% up to 10% dextran sulfate (see Note 5); 0.1% SDS; 0.1% BSA; 1% up to 2% blocking solution; 250 µg/ mL fish sperm DNA (Roche Diagnostic GmbH, Mannheim, Germany); 1–10 ng/µL hybridization probe. Additionally for RT-IS-PCR: 47. DNAse/RNAse solution: 40 mM Tris-HCl, pH 7.4, 6 mM MgCl 2 , 2 mM CaCl 2 , 1 U/µL RNAse free DNAse (Roche Diagnostic GmbH, Mannheim, Germany), and500 µg/mL RNAse (Roche Diagnostic GmbH, Mannheim, Germany). 48. Reverse transcription buffer (RT-buffer): 50 mM Tris-HCl, pH 8.3, 75 mM KCl, 3 mM MgCl 2 , 0.5 mM dNTPs (Roche Diagnostic GmbH, Mannheim, Germany), 10 mM DTT (GibcoBRL, Karlsruhe, Germany), 1 U/µL RNAsin (Promega, Mannheim, Germany), 2 U/µL M-MLV-RT (GibcoBRL, Karlsruhe, Germany) (see Note 6), 0.1 µg/µL Random Hexamers (Roche Diagnostic GmbH, Mannheim, Germany). 3. Methods If not otherwise mentioned, all incubations are performed at ambient temperature (20°C). Incubations on the thermocycler are performed using the humidity chamber. Samples are processed without coverslips and sealing if not otherwise mentioned. 3.1. Prevention of DNAse and RNAse Contamination 1. Incubate all slides, coverslips and microtome blades for 12 h at 220°C to inactivate DNAse and RNAse (see Notes 7 and 8). 2. If RNA detection is required, include 0.1% DEPC in all solutions and incubate overnight. 3. Autoclave solutions for 20 min at 1.2 bar (15 psi) at 121°C. 3.2. Fixation Incubate tissues for 24 h in 5% buffered formalin (for detection of DNA) or 4% buffered paraformaldehyde (for detection of mRNA) (see Note 9). With cell suspensions and cytospins a 30 min incubation is usually sufficient. 3.3. Paraffin Embedding The distilled water used to dilute ETOH should be autoclaved and prepared with DEPC-treated water to ensure the absence of DNAse and RNAse. 1. Incubate tissue for 30–60 min in 70% ETOH. 2. Incubate tissue for 30–60 min in 80% ETOH.
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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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222 Bartlett Notwithstanding these
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224 Bartlett
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226 Dominguez et al. Table 1 Primer
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228 Dominguez et al. 4. Oligonucleo
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230 Dominguez et al. Fig. 2. cDNA n
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232 Dominguez et al. 3.4. Gel Elect
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234 Dominguez et al. 3. If the cont
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236 Dominguez et al. 11. Joshi, C.
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238 Khalturin, Kuznetsov, and Bosch
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246 Ringquist et al. In this chapte
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248 Ringquist et al. 5. Total RNA s
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250 Ringquist et al. 3. Purificatio
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252 Ringquist et al. 2. The present
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254 Ringquist et al.
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256 Case-Green, Pritchard, and Sout
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272 Oien Fig. 1. A schematic diagra
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274 Oien 4. 100% and 70% ethanol. 5
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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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284 Oien
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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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300 Rithidech and Dunn
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302 Edwards and Bartlett Studies th
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304 Edwards and Bartlett 11. Hot st
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306 Edwards and Bartlett Fig. 1. Ex
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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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314 Schmerer
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316 Aubele and Smida 2.2. Chemicals
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318 Aubele and Smida References 1.
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320 Nakashima, Akahoshi, and Tanaka
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322 Nakashima, Akahoshi, and Tanaka
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324 Stirling 9. TAE (20×): 484 g o
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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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Page 445 and 446: 456 Speel, Ramaekers, and Hopman Fi
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Page 457 and 458: 470 Wang 2. Materials 2.1. PCR 1. D
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Page 461 and 462: 474 Wang
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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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