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Day 3: Isolation of Total RNA

Discussion Topics

Isolation of RNA

Experimental Procedures

RNA isolation
Spectrophotometric measurements of purified RNA

Overview of Procedures

Each team will isolate total RNA from Arabidopsis. To confirm the quality and quantity of the isolated RNA, you will a) measure absorbance at 260, 280, and 230 nm, b) determine A260/A280 and A260/A230 ratios, and c) calculate the concentration.

Notes on Molecular Biological Procedures

General Guidelines

Maintain a clean work area
Use a fresh pipet tip for every transfer (tips should be DNase/RNase free)
Wear gloves to prevent contamination (of yourself as well as your experiment)
Sterilize solids and liquids by autoclaving 20 minutes at 121°C at 15 psi

Pipetting Small Volumes

Before beginning the procedure, thaw all frozen reagents and mix well
Pulse spin ALL tubes of aliquots to bring the liquid to the bottom of the tube -- in the microcentrifuge hold the "SHORT" key for about 5-10 seconds
(Capless 1.5 ml vials serve as holders for 0.2 and 0.5 ml tubes in the microcentrifuge rotors)
Touch only the very tip to the surface of the solution (i.e., do NOT submerge the pipet tip into the solution)
Most enzyme stocks are in 50% glycerol; these solutions are quite viscous and liquid will stick to the outside of the pipet tip so touch only the surface

Centrifugation

DO NOT PUT TAPE ON TUBES!
ALWAYS balance the load in the centrifuge
Capless 1.5 ml vials serve as holders for 0.2 and 0.5 ml tubes in the rotors
Pulse spin ALL tubes of aliquots to bring the liquid to the bottom of the tube -- in the microcentrifuge hold the "SHORT" key for about 5-10 seconds
DO NOT SLAM THE LIDS! (this action breaks the latch mechanisms)

Isolation of Total RNA from Arabidopsis thaliana

High quality RNA is critical to the success of qPCR. Degraded or contaminated RNA cannot be efficiently reverse transcribed and, therefore, will not yield good amplification products.

Total RNA will be isolated from Arabidopsis using Qiagen RNeasy Plant Mini Kit.
Tissue will be snap-frozen in liquid nitrogen; you will use a mortar and pestle to grind the frozen tissue. NOTE: a link to the instruction manual from the manufacturer is in Canvas. Since qPCR is sensitive to DNA contamination, we will also use RNase-Free DNase (Qiagen) for an on-column DNase digestion.
The concentration of RNA will be determined by measuring the absorbance at 260 nm; A260/A280 and A260/A230 ratios will be determined to give an estimate of the purity of RNA with respect to protein and organic contaminants, respectively.

Copyright, Acknowledgements, and Intended Use
Created by B. Beason (bbeason@rice.edu), Rice University, 25 June 1999
Updated 10 March 2016