RNA Methodologies. A Laboratory Guide for Isolation and by Robert E. Farrell Jr.

By Robert E. Farrell Jr.

This publication is a set of attempted and validated laboratory protocols for the isolation and characterization of mammalian RNA. It reviews mobile law utilizing RNA as a parameter of gene show, bargains RNA isolation options, and explains right dealing with, garage, and manipulation of RNA.

* stories mobile law utilizing RNA as a parameter of Gene Expression
* bargains RNA isolation strategies
* Explains right dealing with, garage, and manipulation of RNA

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Additional info for RNA Methodologies. A Laboratory Guide for Isolation and Characterization

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At autoclaving temperature and pressure, DEPC degrades into carbon dioxide and ethanol, under which conditions both products are quite volatile. , 1980). Purging can be further promoted by rapidly stirring the hot solutions with a nuclease-free magnetic stir bar. Frequently, one notices the distinctive odor of residual DEPC if autoclaving time was not adequate. In this laboratory, solutions are routinely kept in a warm autoclave for a few hours after completion of the autoclaving cycle, in order to purge them of DEPC.

Preparation of Equipment and Reagents Rule number one when working with RNA is to wear gloves during the preparation of reagents and equipment and especially during the actual RNA extraction procedure. Finger greases, especially those of overly zealous lab technicians, are notoriously rich in RNase and are generally accepted as the single greatest potential source of RNase contamination. P r e p a r a t i ono fEquipmen tan d R e a g e n t s 37 Further, one should not hesitate to change gloves several times during the course of an RNA-related experiment, since door knobs, micropipettors, and refrigerator door handles are also potential sources of nuclease contamination.

The electrophoresis unit itself, including the gel comb, casting tray, and interior of the gel box can be treated to remove RNase con­ tamination by rinsing and soaking in DEPC-treated water (after autoclaving the water). Never expose the unit to DEPC because acrylic is not resistant to DEPC. Many RNases, including the particularly resilient pancreatic variety, manage to renature following treatment and removal of most denaturing reagents. It is prudent to maintain separate containers of chemicals as well as stock solutions for exclusive use as RNA reagents; chemical solids should be weighed out with an RNase-free spatula.

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