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RNase Inhibitor

RiboLock™ RNase Inhibitor

- Thermal inactivation at 75°C in 10 min
- Recombinant enzyme
- LO certified

Catalog#	Size, concentration	Certificate of Analysis	MSDS
Catalog#	Size, concentration	Certificate of Analysis	MSDS
EO0381	2500 u (40 u/µl)	EO0381
EO0382	4 x 2500 u (40 u/µl)	EO0382
EO0384	24 x 2500 u (40 u/µl)	EO0384

Product information

Features

Performs under a wide range of reaction conditions.
Protects RNA from degradation at temperatures up to 55°C.

Applications

Inhibition of RNA degradation in the following:
– in vitro transcription (1),
– cDNA synthesis (2),
– in vitro translation (3),
– isolation of mammalian cell fractions that contain mRNA-protein complex (3),
– RNA amplification (4).
RNA purification and storage.
Separation and identification of specific ribonuclease activities (5).
Studies of tumor suppression (6).

Description

RiboLock™ RNase Inhibitor inhibits the activity of RNases A (see Fig.1), B and C by binding them in a noncompetitive mode at a 1:1 ratio. It does not inhibit eukaryotic RNases T1, T2, U1, U2, CL3 as well as prokaryotic RNases I and H.

Source

E.coli cells with a cloned gene encoding mammalian ribonuclease inhibitor.

Molecular Weight

49.6 kDa monomer.

Definition of Activity Unit

One unit of the RiboLock™ RNase Inhibitor inhibits the activity of 5 ng RNase A by 50%.

Inhibitor activity is assayed in the following mixture: 100 mM Tris-HCl (pH 7.5), 1.2 mM EDTA, 0.1 mg/ml BSA, 100 ng/ml RNase A, 0.1 mg/ml [³H]-RNA, 50 mg/ml yeast RNA, 8 mM DTT.

Storage Buffer

The enzyme is supplied in:
20 mM HEPES-NaOH (pH 7.5), 50 mM NaCl, 8 mM DTT, 0.5 mM ELUGENT Detergent and 50% (v/v) glycerol.

Quality Control

The absence of ribonucleases, endo-, exodeoxyribonucleases and phosphatases and ribonucleases confirmed by appropriate quality tests. Functionally tested in RNA and cDNA synthesis.

Inhibition and Inactivation

Inhibitors: common denaturants (SDS, urea and all oxidizing reagents (p-chloromercuribenzoate, dissolved oxygen, ions in their higher oxidation states) strongly inhibit RiboLock™ RNase Inhibitor and release the RNase bound.
Inactivated by heating at 75°C for 10 min. Residual activity detectable after 10 min heating at 70°C.

Note

DTT provided in the Storage Buffer ensures stability during long term storage, but is not necessary for inhibitor activity.
Recommended concentration 1 u/µl of a reaction mixture.

Figure 1. Inhibition of RNase A by RiboLock™ RNase Inhibitor.
Total human RNA (1 µg) was incubated at 37°C with 20 u of RiboLock™ RNase Inhibitor and increasing amounts of RNase A.
M – RiboRuler™ High Range RNA Ladder
C1 – total human RNA
C2 – total human RNA with RNase A (no RiboLock™ added)
1-4 – total human RNA with RiboLock™ and RNase A

Figure 2. Thermostability of RiboLock™ RNase Inhibitor.
Total human RNA (1 µg) was incubated with 20 u of RiboLock™ RNase Inhibitor and 50 pg of RNase A, and incubated at increasing temperatures.
M – RiboRuler™ High Range RNA Ladder
C1 – total human RNA
C2 – total human RNA with RNase A (no RiboLock™ added)
1-5 – total human RNA with RiboLock™ and RNase A

Patents, Licenses, Trademarks

Protocols & recommendations

RECOMMENDATIONS FOR USE

ADDITIONAL PROTOCOLS

Guidelines to Avoid RNase Contamination

RNA purity and integrity is essential for synthesis of full-length cDNA, which results in high quality RT-PCR products. Therefore, RNase contamination is always a concern when working with RNA. The RNA quality can be affected by RNase A, which is a highly stable contaminant of any laboratory environment. All components of the kit have been rigorously tested to ensure that they are RNase free. To prevent contamination both the laboratory environment and all prepared solutions must be free of RNases.

DEPC-treat all tubes and pipette tips to be used in the cDNA synthesis or use certified nuclease-free labware.
Use pipettes dedicated for RNA work.
Wear gloves when handling RNA and all reagents, as skin is an common source of RNases. Change gloves frequently.
Use certified reagents, including high quality water (e.g., nuclease-free or DEPC-treated Water).
Use an RNase inhibitor, such as RiboLock™ RNase Inhibitor, to protect template RNA.
Always assess the integrity of RNA prior to cDNA synthesis. For example, if sharp bands of both the human 18S rRNA (runs at approx. 1.9 kb) and the 28S rRNA (runs at approx. 5 kb) are formed during denaturing agarose gel electrophoresis of total RNA, the mRNA in the sample is considered to be intact.

I. First Strand cDNA Synthesis

The following protocol is provided for first strand cDNA synthesis using RevertAid™ H Minus Reverse Transcriptase.

All Fermentas reverse transcriptases (RT) are suitable for the synthesis of full-length first strand cDNA, but they differ in reaction temperatures, amounts of RNA transcribed, sensitivity and RNase H activity. The table below indicates reaction conditions recommended for each RT. Enzyme units and RNA amounts are provided for 20 µl of RT reaction volume:

Reverse transcriptase	Reaction temp.	Active up to	Reading length	RHase H activity	Inactivation	Units	Total RNA	poly(A) RNA
Maxima® RT	50-55°C	60°C	20kb	+	85°C, 5min	200	10pg-5µg	0.1pg-500ng
RevertAid™ Premium RT	50-55°C	60°C	20kb	–	85°C, 5min	200	10pg-5µg	1pg-500ng
RevertAid™ H Minus RT	42-45°C	55°C	13 kb	–	70°C, 10min	200	0.1ng-5µg	10pg-500ng
RevertAid™ RT	42°C	50°C	13kb	+	70°C, 10min	200	0.1ng-5µg	10pg-500ng
M-MuLV RT	37°C	37°C	9kb	+	70°C, 10min	40	100ng-5µg	10-500ng
AMV RT	45-60°C	60°C	13kb	++	85°C, 5min	10	10ng-5µg	1-100ng

Master Mix. To prepare several parallel reactions and to minimize the possibility of pipetting errors and contamination, prepare a RT master mix by adding all reaction components except RNA into one vial. Prepare enough master mix for the number of reactions and add one extra to compensate for pipetting errors. Sample template RNA into individual tubes and keep on ice. Aliquote the prepared master mix into tubes with RNA.
Mix and briefly centrifuge all components after thawing, keep on ice.

Add into sterile, nuclease-free tube on ice in the order given:

Total volume	11.5 µl
poly(A)⁺ mRNA or	1 µg
Total RNA or	5 µg
Specific RNA transcript	0.5-1 µg
Oligo(dT)₁₈ primer (0.5 µg/µl) or	0.5 µg
Random hexamer primer (0.2 µg/µl) or	0.2 µg
Gene-specific primer	100 pmol
DEPC-treated Water	to 11.5 µl

Optional (if RNA template is GC-rich or is known to contain secondary structures). Mix gently and briefly centrifuge, incubate at 65°C for 5 min, chill on ice and briefly centrifuge, then place the tube on ice.

Add in the order given:

Total volume	20 µl
5X reaction buffer for reverse transcriptase	4 µl
RiboLock™ RNase Inhibitor	0.5 µl (20 u)
dNTP Mix, 10 mM each	2 µl (1 mM final concentration)
RevertAid™ H Minus Reverse Transcriptase	1 µl (200 u)

Mix gently and briefly centrifuge.
If oligo(dT)₁₈ primer or gene-specific primer is used, incubate 60 min at 42°C.
If random hexamer primer is used, incubate 10 min at 25°C followed by 60 min at 42°C. For reverse transcription of GC-rich RNA reaction temperature can be increased up to 45°C.
Terminate the reaction by heating at 70°C for 10 min.

The reverse transcription reaction product can be directly used in second strand cDNA synthesis or stored at -20°C.

Conventional in vitro Transcription

More than 10 µg of RNA transcript can be generated per 1 µg template DNA using the following protocol. The reaction can be scaled up or down. For high yield transcription, generating up to 200 µg RNA, use TranscriptAid™ T7 High Yield Transcription Kit.

Thaw frozen reagents, mix and centrifuge briefly.
Keep enzymes and nucleotides on ice.
Keep the Reaction Buffer at room temperature.

Prepare the following reaction mixture at room temperature:

Total volume	50 µl
5X Transcription buffer	10 µl
ATP/GTP/CTP/UTP Mix, 10 mM each	10 µl (2 mM final concentration)
Linearized template DNA	1 µg
RiboLock™ RNase Inhibitor	1.25 µl (50 u)
T7/T3/SP6 RNA Polymerase	1.5 µl (30 u)
DEPC-treated Water	to 50 µl

Incubate at 37°C for 2 hours.
Optional: To remove template DNA add 2 µl (2 u) of DNase I, RNase-free, mix and incubate at 37°C for 15 min.
Stop the reaction by addition of 2 µl 0.5 M EDTA, pH 8.0 and incubate at 65°C for 10 min.

Note

RNA hydrolyzes if heated in the absence of a chelating agent.

Synthesis of Radiolabeled RNA Probes of High Specific Activity

Linearize template DNA with a restriction enzyme. Extract DNA with phenol/chloroform, then with chloroform/isoamyl alcohol, and precipitate with ethanol. Dissolve DNA in DEPC-treated Water.

Combine the following reaction components at room temperature in the order given:

Total volume	20 µl
5X Transcription buffer	4 µl
3 NTP Mix, 10 mM each* (without labeled NTP)	1 µl (0.5 mM final concentration)
100 µM CTP	2.4 µl (12 µM final concentration)
[alpha-³²P]-CTP, ~30 TBq/mmol (800 Ci/mmol)	1.85 MBq (50 µCi)
Linear template DNA	0.2-1.0 µg
RiboLock™ RNase Inhibitor	0.5 µl (20 u)
T7/T3/SP6 RNA Polymerase	1 µl (20 u)
DEPC-treated Water	to 20 µl

Incubate at 37°C for 2 hours.
Stop the reaction by cooling at -20°C.
Determine the percentage of the label incorporated into RNA.

Note

* To prepare a mix of the three non-labeled NTPs 10 mM each, combine 1 µl of all three NTPs, 100 mM, from the set (#R0481) with 7 µl of DEPC-treated Water. Store the mix at -20°C for further use.

Expect specific radioactivity of 3-5 x10⁸ dpm/µg.
RNA can be radiolabeled with [³²P], [³⁵S] or [³H]-ribonucleotides. Recommended amounts of radiolabeled nucleotides in 20 µl of reaction mixture are as follows: 1.85 MBq (50 µCi) for 5'-[alpha-³²P]-CTP, approx. 30 TBq/mmol (800 Ci/mmol); 11.1 MBq (300 µCi) for 5'-[alpha-³⁵S]-UTP, more than 37 TBq/mmol (1000 Ci/mmol); 0.925 MBq (25 µCi) for 5,6-[³H]-UTP, 1.1-2.2 TBq/mmol (30-60 Ci/mmol).
The yield of the full-length transcripts is reduced when the concentration of labeled NTP is below 12 µM.

Radioactive Labeling of RiboRuler™ RNA Ladders by T4 Polynucleotide Kinase

The ready-to-use versions of RiboRuler™ RNA ladders can not be radiolabeled with T4 Polynucleotide Kinase.
For efficient labeling of RNA ladders it is recommended to remove 5'-phosphate groups from RNA and then phosphorylate in forward reaction using T4 Polynucleotide Kinase.
I. Dephosphorylation

Prepare the following reaction mixture:

Total volume	20 µl
RiboRuler™ Low Range RNA Ladder or RiboRuler™ High Range RNA Ladder	8 µl
RiboLock™ RNase Inhibitor	0.5 µl (20 u)
10X reaction buffer for alkaline phosphatase	2 µl
FastAP™ Thermosensitive Alkaline Phosphatase or Shrimp Alkaline Phosphatase	2 µl (2 u)
DEPC-treated Water	to 20 µl

Incubate at 37°C for 30 minutes.
Remove proteins from the mixture with a 20 µl aliquot of Tris-saturated (pH 8.0) phenol and chloroform mixture. Save the upper aqueous phase and extract it twice with 20 µl aliquot of chloroform.
Precipitate RNA by adding 1 µl of 3 M Sodium Acetate Solution, 55 µl of 96% ethanol and keep 15-30 min at -20°C. Centrifuge the mixture for 20 min at 10,000-15,000 rpm at 4°C.
Rinse the pellet with 20 µl cold 75% ethanol. Centrifuge 10 min at 10,000-15,000 rpm, 4°C.
Discard the supernatant and dissolve the air-dried pellet in 10 µl of DEPC-treated Water.

II. Labeling

Prepare the following reaction mixture:

Total volume	10 µl
Dephosphorylated RiboRuler™ Low Range RNA Ladder or Dephosphorylated RiboRuler™ High Range RNA Ladder	1 µl 2.5 µl
[gamma-³²P]-ATP (5000 Ci/mmol,10 µCi/µl)*	5 µl (10 pmol)
RiboLock™ RNase Inhibitor	0.25 µl (10 u)
10X buffer A for forward reaction (supplied with T4 polynucleotide kinase)	1 µl
T4 Polynucleotide Kinase	1 µl (10 u)
DEPC-treated Water	to 10 µl

* If [gamma-³²P]-ATP with a high specific activity (higher than 5000 Ci/mmol) is used, the label can be diluted with ATP. Total ATP concentration should be at least 1 µM.

Incubate at 37°C for 30 minutes.
Stop the reaction by adding 1 µl of 0.5 M EDTA, pH 8.0 and extract the mixture with an equal volume of chloroform.
Determine the efficiency of label incorporation.
Load the ladder on the gel.

Synthesis of cDNA Probes with High Specific Radioactivity and Non-radioactively Labeled cDNA

I. Synthesis of cDNA probes with high specific radioactivity

This protocol is provided for first strand cDNA synthesis using RevertAid™ H Minus Reverse Transcriptase. For specific reaction conditions using other enzymes, see Table below. Enzyme units and RNA amounts are provided for 20 µl of RT reaction volume:

Reverse transcriptase	Reaction tmp.	Active up to	Reading length	RHase H activity	Inactivation	Units	Total RNA	poly(A) RNA
Maxima® RT	50-55°C	60°C	20kb	+	85°C, 5min	200	1pg-5µg	0.1pg-500ng
RevertAid™ Premium RT	50-55°C	60°C	20kb	–	85°C, 5min	200	1pg-5µg	0.1pg-500ng
RevertAid™ H Minus RT	42-45°C	55°C	13 kb	–	70°C, 10min	200	0.1ng-5µg	10pg-500ng
RevertAid™ RT	42°C	50°C	13kb	+	70°C, 10min	200	0.1ng-5µg	10pg-500ng
M-MuLV RT	37°C	37°C	9kb	+	70°C, 10min	40	100ng-5µg	10-500ng
AMV RT	45-60°C	60°C	13kb	++	85°C, 5min	10	10ng-5µg	1-100ng

Mix and briefly centrifuge all components after thawing, keep on ce.

Add into sterile, nuclease-free tube on ice in the order given:

Total volume		8.5 µl
Template RNA	Total RNA or	up to 5 µg
	Poly(A) RNA or	up to 500 ng
	Specific RNA or	up to 500 ng
Primers	Oligo(dT)₁₈ or	0.5 µg (100 pmol)
Primers	Random Hexamer	0.2 µg (100 pmol)
Gene-specific		15-20 pmol
DEPC-treated Water		to 8.5 µl

Mix gently and centrifuge to collect all drops.
Optional. Incubate at 65°C for 5 min, chill on ice and briefly centrifuge to collect drops. Perform this step if RNA template is GC-rich or is known to contain secondary structures.

Place the tube with primer/template mix on ice and add the following components in the indicated order:

Total volume	20 µl
5X reaction buffer	4 µl
RiboLock™ RNase Inhibitor	0.5 µl (20 u)
dGTP, dCTP, dTTP mix, 10 mM each	1 µl
0.1 mM dATP	4 µl
[alpha-³²P]-dATP, 3000 Ci/mmol	1 µl
RevertAid™ H Minus Reverse Transcriptase	1 µl (200 u)

Mix gently and centrifuge to collect all drops.
If Oligo(dT)₁₈ primer or gene-specific primer is used, incubate 60 min at 42°C. If random hexamer primer is used, incubate 10 min at 25°C followed by 60 min at 42°C. For transcription of GC-rich RNA reaction temperature can be increased to 45°C.
Stop the reaction by adding 5 µl of 0.5 M EDTA, pH 8.0.
Optional. Hydrolyze RNA by the addition of equal volume (25 µl) of 0.6 M NaOH and incubation at 70°C for 30 min.
Remove unincorporated dNTPs by chromatography on a Sephadex® G-50 column.
Expect specific radioactivity of >10⁷ dpm/µg.

Note

To achieve higher specific activities (over 10⁸ dpm/µg), use up to 100 µCi of [alpha-³²P]-dATP in the labeling mixture. To keep the total reaction volume of 20 µl, vacuum-evaporate 10 µl of [alpha-³²P]-dATP (10 mCi/ml) to 1 µl in a separate tube.

II. Synthesis of non-radioactively labeled cDNA

The same protocol can be used for synthesis of non-radioactive labeled cDNA using biotin-11-dUTP, fluorescein-12-dUTP, DIG-dUTP or aminoallyl-dUTP:

normal dTTP is subsituted with labeled-dUTP at a molar ratio of 1:3-1:4,
reaction time is prolonged to 2-6 hours.

Removal of Genomic DNA from RNA Preparations

Add to an RNase-free tube:

RNA 1 µg

10X reaction buffer with MgCl₂ 1 µl

DNase I, RNase-free 1 µl (1 u)

DEPC-treated Water to 10 µl

Total volume 10 µl
Incubate 30 min at 37°C.
Add 1 µl of 50 mM EDTA and incubate 10 min at 65°C. RNA hydrolyzes if heated in the absence of a chelating agent (1).
Use the prepared RNA as a template for reverse transcriptase.

Note

Do not use more than 1 u of DNase I, RNase-free per µg of RNA.
Reaction mixture can be scaled up for larger amounts of RNA. The recommended final concentration of RNA is 0.1-0.2 µg/µl.
RiboLock™ RNase Inhibitor (#EO0381), typically at 1 u/µl, can also be included in the reaction mixture to inactivate type A RNases potentially present in the initial RNA solution

Reference

Wiame, I., et al., Irreversible heat inactivation of DNase I without RNA degradation, BioTechniques, 29, 252-256, 2000.

References

Nielsen, D.A., Shapiro, D.J., Preparation of capped RNA transcripts using T7 RNA polymerase, Nucleic Acids Res., 14, 5936, 1986.
Martynoff, G., et al., Synthesis of a full length DNA complementary to thyroglobulin 33S messenger RNA, Biochem. Biophys. Res. Commun., 93, 645-653, 1980.
Scheele, G., Blackburn, P., Role of mammalian RNase inhibitor in cell-free protein synthesis, Proc. Natl. Acad. Sci. USA, 76, 1898-1902, 1979.
Van Gelder., et al., Amplified RNA synthesized from limited quantities of heterogeneous cDNA. Proc. Natl. Acad. Sci. USA, 87, 1663-1667, 1990.
Eichler, D.C., et al., Effect of human placental ribonuclease inhibitor in cell-free ribosomal RNA synthesis, Biochem. Biophys. Res. Commun., 101, 396-403, 1981.
Polakowski, I.J., et al., A ribonuclease inhibitor expresses anti-angiogenic properties and leads to reduced tumor growth in mice, Amer. J. Pathol., 143, 507-517, 1993.

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