How Does depc inhibit rnase? 2|3 How Are DEPC-Treated Solutions Prepared? Is or Should You Treat Your Pre-made Reagents with r213 More depc better Should You Prepare Reagents with DEPC-Treated Wate DEPC? ..2|4 How Do You Minimize RNA Degradation during Sample Collection and storage ..2|4 How Do You Minimize RNA Degradation during Sample Disruption? Is There a Safe Place to Pause during an rna Purification Procedure? 2|8 What Are the Options to Quantitate Dilute RNA 2|8 What Are the Options for Storage of Purified RNAS 2l9 Troubleshooting 220 A Pellet of Precipitation RNA Is Not Seen at the End of he rna Purification A Pellet Was Generated, but the Spectrophotometer Reported a Lower Reading Than Expected, or Zero Absorbance RNA Was Prepared in Large Quantity, but in a downstream Reaction RT PCr is Example My Total RNA Appeared as a Smear in an Ethidum Bromide-stained Denaturing Agarose Gel; 18S and 28S RNA Bands Were not observed 222 Only a Fraction of the Original RNA Stored at-70C Remained after Storage for Six Months 222 Bibliography SELECTING A PURIFICATION STRATEGY Do Your Experiments Require Total RNA or mRNA? One of the first decisions that the researcher has to make when detecting or quantitating RNA is whether to isolate total RNA or poly (A)-selected RNa (also commonly referred to as mRNA) This choice is further complicated by the bewildering array of RNA isolation kits available in the marketplace. In addition the downstream application influences this choice. The following section is a short primer in helping make that decision From a purely application point of view, total RNA might suffice for most applications, and it is frequently the starting material for applications ranging from the detection of an mRNA species by Northern hybridization to quantitation of a message by Martin et alHow Does DEPC Inhibit RNase? . . . . . . . . . . . . . . . . . . . . . . 213 How Are DEPC-Treated Solutions Prepared? Is More DEPC Better? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 213 Should You Prepare Reagents with DEPC-Treated Water, or Should You Treat Your Pre-made Reagents with DEPC? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 214 How Do You Minimize RNA Degradation during Sample Collection and Storage? . . . . . . . . . . . . . . . . . . . . . . . . . . . . 214 How Do You Minimize RNA Degradation during Sample Disruption? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 215 Is There a Safe Place to Pause during an RNA Purification Procedure? . . . . . . . . . . . . . . . . . . . . . . . . . . . . 218 What Are the Options to Quantitate Dilute RNA Solutions? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 218 What Are the Options for Storage of Purified RNA? . . . . . . . 219 Troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 220 A Pellet of Precipitation RNA Is Not Seen at the End of the RNA Purification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 220 A Pellet Was Generated, but the Spectrophotometer Reported a Lower Reading Than Expected, or Zero Absorbance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 221 RNA Was Prepared in Large Quantity, but it Failed in a Downstream Reaction: RT PCR is an Example . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 221 My Total RNA Appeared as a Smear in an Ethidum Bromide-stained Denaturing Agarose Gel; 18S and 28S RNA Bands Were not Observed . . . . . . . . . . . . . . . . 222 Only a Fraction of the Original RNA Stored at -70°C Remained after Storage for Six Months . . . . . . . . . . . . . . 222 Bibliography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 222 SELECTING A PURIFICATION STRATEGY Do Your Experiments Require Total RNA or mRNA? One of the first decisions that the researcher has to make when detecting or quantitating RNA is whether to isolate total RNA or poly(A)-selected RNA (also commonly referred to as mRNA). This choice is further complicated by the bewildering array of RNA isolation kits available in the marketplace. In addition the downstream application influences this choice. The following section is a short primer in helping make that decision. From a purely application point of view, total RNA might suffice for most applications, and it is frequently the starting material for applications ranging from the detection of an mRNA species by Northern hybridization to quantitation of a message by 198 Martin et al