Search Thermo Fisher Scientific
Search Thermo Fisher Scientific
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
Caution
General Guidelines
Problem | Cause | Solution |
---|---|---|
Low RNA yield | Incomplete lysis and homogenization |
|
Poor quality of starting material |
| |
Clogged RNA Spin Cartridge | Clear homogenate and remove any particulate or viscous material by centrifugation, and use only the supernatant for subsequent loading onto the Spin Cartridge. | |
Ethanol not added to Wash Buffer II | Add ethanol to Wash Buffer II before use. | |
Incorrect elution conditions | Add RNase–Free Water (30–3 ×100 μl) and perform incubation for 1 minute before centrifugation. To recover more RNA, be sure to use up to 3 sequential elutions of 100 μl each (3 × 100 μl) Elution Buffer (refer to Elution protocol. | |
RNA degraded | RNA contaminated with RNase | Follow the guidelines above to prevent RNase contamination. |
Improper handling of sample from harvest until lysis | If you are not processing your tissue samples immediately after harvest, quick–freeze tissue immediately after harvesting and store at –80°C or in liquid nitrogen. Keep the samples frozen until TRIzol® Reagent is added. Perform the lysis quickly after adding TRIzol® Reagent. | |
Inhibition of downstream enzymatic reactions | Presence of ethanol in purified RNA | Traces of ethanol from the Wash Buffer II can inhibit downstream enzymatic reactions. Discard Wash Buffer II flow through. Place the Spin Cartridge into the Recovery Tube and centrifuge at 12,000 × g for 1-2 minutes to completely dry the cartridge |
Presence of salt in purified RNA | Use the correct order of Wash Buffers for washing. Always wash with Wash Buffer I followed by washing with Wash Buffer II. | |
Low A260/A280 ratio | Sample was diluted in water | Use 10 mM Tris–HCl (pH 7.5) to dilute sample for OD measurements. |