TRIzol Plus RNA Purification Kit

Description

The TRIzol Plus RNA Purification Kit provides a simple, reliable, and rapid method for isolating high–quality total RNA from a wide variety of samples, including animal and plant cells and tissue, bacteria, and yeast. The kit utilizes the strong lysis capability of TRIzol Reagent, followed by a convenient and time–saving silica–cartridge purification protocol from the PureLink™ RNA Mini Kit, to purify ultrapure total RNA within an hour, even from difficult samples such as fibrous tissue. TRIzol Reagent is a monophasic solution of phenol, guanidine isothiocyanate, and other proprietary components which facilitate the isolation of a variety of RNA species of large or small molecular size. TRIzol Reagent maintains the integrity of the RNA, while disrupting cells and dissolving cell components, and also provides an immediate and highly effective inhibition of RNase activity during sample homogenization or lysis.

The combination of TRIzol Reagent followed by silica–cartridge purification of RNA is the recommended RNA purification method in the gene expression industry. The TRIzol Plus RNA Purification Kit provides the reagents and an optimized protocol to purify total RNA for gene expression studies using an industry recommended method.

System Overview

To isolate purified RNA from your sample using the TRIzol Plus RNA Purification Kit, your sample is first lysed with TRIzol Reagent, according to the lysate preparation protocol provided. The addition of chloroform to your sample, followed by centrifugation separates the solution into an upper aqueous phase containing RNA and an lower phenol-containing organic phase. The upper aqueous phase is transferred to a new tube, followed by ethanol addition and centrifugation. The sample is then transferred to the PureLink™ RNA Mini Kit Spin Cartridge containing a clear silica–based membrane to which the RNA binds during purification. The RNA is washed to remove contaminants and the purified total RNA is then eluted in RNase–Free Water (Tris Buffer, pH 7.5 may also be used) and is suitable for use in a variety of downstream applications including sensitive gene expression studies such as microarray analysis or real time quantitative RT-PCR (qRT-PCR).

PureLink™ RNA Mini Kit Specifications

Cartridge Binding Capacity: ~1 mg nucleic acid
Cartridge Reservoir Capacity: 700 μl
Wash Tube Capacity: 2.0 ml
Centrifuge Compatibility: Capable of centrifuging >12,000 × g
Elution Volume: 30 μl–3 × 100 μl (3 sequential elutions with 100 μl each)

Notes

• This page provides instructions for purifying total RNA from samples using TRIzol Reagent to prepare lysates and after phase separation, the RNA is purified using the PureLink™ RNA Mini Kit. If you wish to use other protocols for RNA purification, refer to the TRIzol Reagent manual or the PureLink™ RNA Mini Kit manual.
• The TRIzol Plus RNA Purification Kit utilizes the TRIzol Reagent for sample lysis. The Lysis Buffer included wit  the kit is not used in this protocol. However, if you wish to prepare samples using the Lysis Buffer, refer to the PureLink™ RNA Mini Kit manual.
• If your downstream applications require DNA–free total RNA, protocols for on–column PureLink™ DNase treatment during RNA purification or after purification are provided in the PureLink™ RNA Mini Kit manual.

Caution

• TRIzol Reagent contains phenol (toxic and corrosive) and guanidine isothiocyanate (an irritant), and may be a health hazard if not handled properly. Avoid direct contact with TRIzol Reagent, as direct contact of skin, eyes, or respiratory tract with TRIzol Reagent may cause chemical burns to the exposed area. When working with TRIzol Reagent, always work in a fume hood. Refer to the TRIzol Reagent product insert for more details. Contact your Environmental Heath and Safety (EH&S) department for proper work and disposal guidelines.
• Both Lysis Buffer and Wash Buffer I contain guanidine isothiocyanate (an irritant).
• This chemical is harmful when in contact with the skin, or when it is inhaled or ingested. Do not add bleach or acidic solutions directly to solutions or sample preparation waste that contains guanidinium isothiocyanate, as reactive compounds and toxic gases are formed. Solutions containing ethanol are considered flammable. Use appropriate precautions when using this chemical.
• For your protection, always wear a laboratory coat, gloves and safety glasses when handling these chemicals. Dispose of the buffers and chemicals in appropriate waste containers.

General Guidelines

• The maximum RNA binding capacity of the PureLink™ RNA Mini Kit Spin Cartridge is ∼1 mg. If you are processing samples that contain more than 1 mg of total RNA, divide the sample into aliquots such that each aliquot contains <1 mg total RNA for each Spin Cartridge used.
• Use disposable, individually wrapped, sterile plastic ware and sterile, disposable RNase-free pipette tips and tubes.
• Wear disposable gloves while handling reagents and RNA samples to prevent RNase contamination from the surface of the skin; change gloves frequently, particularly as the protocol progresses from crude extracts to more purified material.
• Always use proper microbiological aseptic techniques when working with RNA.
• Use clear polypropylene disposable tubes when working with <2 ml volumes of TRIzol Reagent. For larger volumes, use glass (Corex) or polypropylene tubes, and ensure that the tubes can withstand centrifugation at 12,000 × g with TRIzol Reagent and chloroform. Do not use tubes that leak or crack.
• Use RNase AWAY Reagent (cat. no. 10328-011) to remove RNase contamination from work surfaces and non– disposable items such as centrifuges and pipettes used during purification.

• Starting material (fresh or frozen tissue, or cells)
• TRIzol Reagent and PureLink™ RNA Mini Kit (included)
• Chloroform or 4–Bromoanisole
• 96-100% ethanol and 70% ethanol (in RNase-free water)
• Microcentrifuge capable of centrifuging at 12,000 × g
• Homogenizer (Cat. no: 12183–026) or Rotor–Stator homogenizer
• 1.5 ml RNase–free microcentrifuge tubes and RNase–free pipette tips