RNA preparation : A number of commercial RNA extraction methods are compatible with microarray analysis. Both solid-phase and chaotropic-salt methods give acceptable results. For solid-phase methods we recommend purification kits such as Qiagen RNeasy or Stratagene Absolutely RNA. In experienced hands these give clean uncontaminated RNA, but RNA degradation can occur if endogenous RNases are not inactivated quickly. Chaotropic-salt purification methods have the advantage that the initial total RNA is less degraded, but extra precautions have to be taken to prevent carryover of phenol and guanidinium salts that will inhibit the labeling reaction. At a minimum this entails re-precipitation of the RNA using sodium acetate/ethanol or further cleanup on a solid phase column. Of the commercial reagents tested, RNABee , Trizol , and Ultraspec have given good results. Genomic DNA contamination is possible with these methods if the phase interface is disturbed. In this case, it is advisable to dissolve the RNA in the reagent a second time and re-extract making sure to avoid the interface. Purified total RNA should be resuspended in RNase-free TE0.1 (10 mM Tris.Hcl, pH 8, 0.1 mM EDTA) at the desired concentration.
Tissues vary somewhat in RNA content and yield. A general rule of thumb is to estimate that one cell will yield 10 pg total RNA. This implies that half a million cells will yield 5 µg. For solid cellular tissues, 10-30 mg will yield sufficient RNA in most cases. The yield from less cellular tissues such as cartilage and bone varies greatly according to technique.
DNA Preparation : Genomic DNA should be isolated using the Proteinase K/SDS method or using a commercial kit. It is important that the DNA be free of phenol contamination. DNA should be resuspended in TE0.1 (10 mM Tris.HCl, pH 8, 0.1 mM EDTA) at the desired concentration. Two micrograms of human genomic DNA corresponds to approximately 3 x 105 cells.
Chromatin immunoprecipitation : High quality immunoprecipitated chromatin DNA, amplified by ligation mediated PCR (LM-PCR) is required. We recommend Nimblegen’s Chromatin Immunoprecipitation & Amplification Protocol for DNA preparation. Please click on the link to view and print the pdf version of the protocol.
Chromatin Immunoprecipitation and Amplification Protocol 
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