Sigma-Aldrich Protector Rnase Inhibitor

Assay

>95% (SDS-PAGE)

Quality Level

100

Mol Wt

~50 kDa

Packaging

pkg of 10,000 U (03335402001 [5 x 2,000 U])
pkg of 2,000 U (03335399001)

Manufacturer/Tradename

Roche

Parameter

25-55 °C optimum reaction temp.

pH Range

5.0-9.0

Storage Temp.

−20°C

Related Categories

PCR Reagents & Kits

Roche® Life Science Products

General Description

Contents
Protector RNase Inhibitor (40 U/μl) in storage buffer: 20 mm HEPES-KOH, 50 mm KCl, 8 mm dithiothreitol, 50% glycerol (v/v), pH approximately 7.6 (+4°C).Protector RNase Inhibitor inactivates a wide spectrum of RNases, including

• RNase A
• RNase B
• RNase T2

Thus, Protector RNase Inhibitor can help prevent RNase degradation in any application where RNases could cause problems. For instance, it can:

• protect mRNA during cDNA synthesis reactions, RT-PCR (in conventional thermal cyclers and qPCR systems), orin vitrotranscription/translation reactions
• protect viral RNA duringin vitrovirus replication
• inhibit RNases during RNA isolation and purification
• be used in RNase protection assays
• help prepare RNase-free antibodies

Note: Protector RNase Inhibitor does not interfere with enzymes commonly used to prepare or analyze RNA.

Specificity

Protector RNase Inhibitor definetely does not inhibit RNase T1 and RNase 1.
Protector RNase Inhibitor (20 U/ 20 μl reaction) inhibits RNase A up to 1 ng, RNase B up to 160 pg and RNase T2 up to 0.03 U/μl reaction volume.
RNase H is not inhibited by Protector RNase Inhibitor.
Heat inactivation:> 65 °C

Application

Protector RNase Inhibitor may be used to:

• Protect mRNA in cDNA synthesis reactions, RT-PCR (in conventional thermal cyclers and qPCR systems),e.g., with the LightCycler^®Instruments,in vitrotranscription/translation system, RNase Protection Assay,in vitroRNA synthesis
• Protect viral RNA duringin vitrovirus replication
• Inhibit RNases during RNA isolation and purification
• Help prepare RNase-free antibodies
• Synthesize RNA probes forin situhybridization[5][6]

It is useful in any application where RNases could be a potential problem.

Features And Benefits

• Rely on a thermostable RNase inhibitor: Protector RNase Inhibitor remains stable even when using thermostable reverse transcriptases such as Transcriptor Reverse Transcriptase.
• Benefit from a wide range of reaction conditions: Protector RNase Inhibitor is active at pH 5.0 to 9.0 and at temperatures between +25°C to +55°C (partial activity is still measureable at +60°C).
• Eliminate interference in different RNA analysis applications: Maintain performance even when adding at 16-fold higher than the standard concentration.
• Insist on a highly-purified preparation: Each batch is function tested using techniques such as quantitative RT-PCR to ensure the absence of endonucleases, ribonucleases, or nicking activity, according to the current quality control procedures.

Quality

Each lot of Protector RNase Inhibitor is function tested with the Titan One Tube RT-PCR Kit and the LightCycler DPD Kit. Protector RNase Inhibitor is also tested for contaminating activities as described below.
Test buffer: 50 mm Tris-HCl, 10 mm MgCl₂, 0.1 mm EDTA, 7 mm β-Mercaptoethanol; pH 7.5 (+37°C).
Absence of endonucleases: 1 μgEcoRI/HindIII*-fragments of λDNA is incubated with Protector RNase Inhibitor in 50 μl test buffer at +37°C for 1 hour. The amount of inhibitor that causes no alteration in the banding pattern is stated under "Endo".
Absence of nicking activity: 1 μg supercoiled pBR322 DNA is incubated with Protector RNase Inhibitor in 50 μl test buffer at +37°C for 1 hour. The amount of inhibitor that causes no relaxation of supercoiled DNA is stated under "Nick. Act.".
Absence of ribonuclease (1): 5 μg of MS2 RNA is incubated with Protector RNase Inhibitor for 1 hour at +37°C in a final volume of 50 μl. The amount of inhibitor that causes no degradation of MS2 RNA is stated under "RNase 1".
Absence of ribonuclease (2): 5 μg of MS2 RNA is incubated with Protector RNase Inhibitor for 1 hour at +37°C, then 10 minutes at +65°C in a final volume of 50 μl. The amount of inhibitor that causes no degradation of MS2 RNA is stated under "RNase 2".

Unit Definition

One unit of Protector RNase Inhibitor is defined as the amount of protein required to inhibit 50% of the activity of 5 ng RNase A.

Unit Assay: Activity is measured according to Blackburn as ability to inhibit hydrolysis of cyclic cytidine-2′ : 3′-monophosphoric acid. Under assay conditions, 200 U of Protector RNase Inhibitor inhibits 50% of the activity of 1 μg RNase A.

Volume Activity: 40 U/μl

Preparation Note

Working concentration:

• 5 to 10 U in One-step RT-PCR
• 25 to 50 U in Two step RT-PCR
• 20 U for in vitro transcription

You may use higher concentrations of Protector RNase Inhibitor in RT-PCR if you suspect that RNase contamination causes certain samples to be difficult to amplify. The inhibitor does not interfere with the reaction. (In a test system, even a 16-fold higher concentration
of inhibitor did not interfere with RT-PCR.)

Other Notes

Product Specifications:
Source: Rat lung; Product is produced recombinant inE. coli
Isoelectric Point: pH 4.5
Temperature where Protector RNase Inhibitor is active: 25°C to +55°C; partial activity is still measurable at +60°C
Inactivation: Under severe denaturizing conditions (temperatures above +65°C), the inhibitor is inactivated
Bioburden: <50 CFU/mL
DNA Content: <100 pg/mgFor life science research only. Not for use in diagnostic procedures.NOTICE TO PURCHASER: DISCLAIMER OF LICENSEThis product is optimized for use in the polymerase chain reaction (PCR) covered by patents owned by F. Hoffmann-La Roche Ltd ("Roche"). No license under these patents to use the PCR Process is conveyed expressly or by implication to the purchaser by the purchase of this product. A license to use the PCR Process for certain research and development activities accompanies the purchase of certain Roche, Applied Biosystems or other licensed suppliers′ reagents when used in conjunction with an authorized thermal cycler, or is available from Applied Biosystems. Diagnostic purposes require a license from Roche. Further information on purchasing licenses may be obtained by contacting the Director of Licensing at Applied Biosystems, 850 Lincoln Centre Drive, Foster City, California 94404, USA.LightCycler is a registered trademark of Roche