RNA Isolation/Extraction

INTRODUCTION

RNA isolation from cells or tissues is the first step in a number of routine laboratory experiments to assess gene expression levels and is used in assays including quantitative PCR, microarray analysis, and Northern blots.  To accurately analyze RNA transcripts, it is important to extract high-quality RNA from the cells or tissues of interest with minimal RNA degradation.  This can be accomplished using the traditional guanidinium thiocyanate–phenol–chloroform extraction method1, a commercial derivative (TriZol®), or any number of commercially available kits.

Regardless of the method used, limiting ribonuclease (RNase) activity is critical to successful RNA isolation and extraction.  Keeping samples cold (4°C) prior to RNA extraction limits RNase activity and safely preserves RNA for accurate analysis of gene transcripts. Commercially available RNA stabilizing reagents can be combined with cold storage to minimize RNA degradation prior to RNA extraction.  This protocol describes how BioCision’s thermo-conductive CoolBox ™ XT and CoolRack® module can be incorporated into a general RNA extraction workflow, and provides specific instructions for extracting RNA from blood, a tissue especially high in RNases, using the TempusTM Kit2 (Life Technologies).

 

MATERIALS NEEDED:

•  CoolBox XT ice-free microfuge tube cooling workstation or, alternatively, CoolRack M15 module with ice pan
•  RNA Extraction Kit of user’s choice. Examples include TriZol and commercially available Spin Kits.  For blood, we refer to the Tempus Blood RNA Tubes and Spin RNA Isolation Kit in this protocol.
•  Vortexer
•  Microcentrifuge
•  Heating block for microcentrifuge tubes
•  Centrifuge (temperature controlled, >3,000 x g)
•  RNase-free microcentrifuge tubes

 

INSTRUCTIONS:

1: Follow manufacturer’s instructions for your preferred RNA Extraction Kit.

  1. A.  Kits and reagents such as TriZol offer some RNA protection.  These protocols are often carried out at room temperature, or a combination of room temperature and 4°C.  Refer to manufacturer’s instruction manual for proper RNA isolation temperature.

  1. B.  Blood is especially high in RNases.  The Tempus Blood RNA Tubes and Spin RNA Isolation Kit can be used in combination with the CoolRack thermo-conductive tube module to minimize RNA degradation.

i.  Following the Tempus Blood RNA Tube instructions and appropriate laboratory safety practices, collect blood into tubes.

ii.  Vortex briefly.

iii.  Store tubes at 4°C in a CoolBox XT ice-free cooling station to decrease the chances of contamination due to ice or in a CoolRack module on ice until ready to proceed.  Maintaining a constant and uniform temperature of 4°C is essential to inhibit RNase activity in all tubes.

iv.  Proceed with manufacturer’s instructions for the Blood RNA Tubes and Isolation Kit.

2: According to manufacturer’s instructions, resuspend RNA in RNase-free water. While working on the bench top, extracted RNA should be maintained at 4°C to prevent RNase degradation.

  1. A.  RNA Quality Control:  While using the spectrophotometer to measure RNA concentration and quality (A260/280) keep the tubes of RNA in a CoolBox XT workstation or CoolRack module, on ice.

  1. B.  Aliquoting: If the isolated RNA is to be used in multiple experiments, aliquoting the sample will prevent RNA degradation due to accidental RNase contamination.  The risk of RNase contamination increases the more frequently a sample is accessed. The CoolBox XT with the CoolRack module can be used to organize tubes while aliquoting and to keep the RNA tubes uniformly cold, thereby preventing RNase activity during the aliquoting process. A constant temperature of 4°C will preserve RNA integrity, resulting in highly reproducible results in downstream applications.

3: For long-term storage, we recommend that RNA isolates be stored at or below -60°C to prevent degradation.

See other protocols:


BioCision RT-PCR protocol
BioCision qPCR protocol
BioCision General PCR Application
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Learn more about:

CoolRack tube modules

CoolBox XT and CoolBox 2XT ice-free cooling workstations

Ice pans and buckets

TruCool ergonomic microfuge tubes

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