RNase R with RNaseOFF Bundle
| Cat. No. | E049-G138S |
| Name | RNase R with RNaseOFF Bundle |
| Unit | Bundle |
| Category | Molecular Biology Enzymes and Kits |
| Description |
Pair one of abm's best-selling products with our high performance RNaseOFF Ribonuclease Inhibitor! RNase R (Cat. No. E049) is an E. coli exoribonuclease that exhibits 3'-to-5' exonuclease activity, efficiently digesting nearly all linear RNA species. This enzyme does not digest circular, lariat, or double-stranded RNA with short 3’ overhangs (less than seven nucleotides). As such, this enzyme is ideally suited to the study of lariat RNA produced by traditional splicing, as well as circRNAs which arise through back-splicing. By removing linear RNAs from cellular or RNA extracts, RNase R greatly facilitates the identification of circular species through RNA-sequencing. This enables researchers to probe the landscape of splicing events with greater depth. To further the protection and enhancement of circRNAs, this bundle includes our high performance RNaseOFF Ribonuclease Inhibitor (Cat. No. G138S) for one special price! This bundle includes:
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| Material Citation | If use of this material results in a scientific publication, please cite the material in the following manner: Applied Biological Materials Inc, Cat. No. E049-G138S |
| Are all components in this product RNase-free? | |
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Yes, all components in the RNaseOFF Ribonuclease Inhibitor is RNase-free.
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| What is the shelf life of this reagent and how should it be stored? | |
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The shelf life of RNaseOFF is 1 year upon shipment of the reagent and it should be stored at -20°C for long-term storage.
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| How many reactions can be performed? | |
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The appropriate number of units per reaction will vary depending on the amount of RNase contamination that is present in the reaction mixture for any given experiment. One unit of RNaseOFF Ribonuclease Inhibitor will inhibit the activity of 5 ng of RNase by 50%; thus, the more RNase contamination present in the specific reaction, the larger number of units of RNaseOFF that will be required for the inhibition. As a general guideline, a typical amount of RNaseOFF used would be around 25-50 U per 50 µl reaction; note that this is a general guideline only and the number of units required will vary from case to case.
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| Is DTT required for the RNaseOFF Ribonuclease Inhibitor to function? | |
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No. DTT (dithiothreitol) is used to stabilize enzymes and proteins, and it is not required for the RNaseOFF Ribonuclease Inhibitor to function/inhibit ribonucleases.
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| Does RNaseOff (Cat. No. G138) inhibit RNase R activity? | |
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No, RNAseOff does not inhibit RNase R activity.
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| How efficiently will RNase R (when best optimized) digest linear RNA? | |
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To calculate the percentage of digested linear RNA in your experiment, perform the following steps: Step 1. Determine the Ct difference (ΔCt) between the Treated (+RNase R) and Untreated (No RNase R) Set. Step 2. Calculate the fold change using the ΔCt value: Fold Change = 2ΔCt Step 3. Calculate the percentage of RNA degraded using the fold change: % RNA Degraded = (1 - (1 / Fold Change)) × 100
For example, if your Treated Set has Ct=28.55 and Untreated Set has Ct = 18.01, the calculation will be: ΔCt = 28.55 – 18.01 = 10.54 Fold Change = 2ΔCt = 210.54 = 1488.87 % RNA Degraded = (1-(1/1488.87)) × 100 = 99.93% |
| What is the length of RNA that can be digested by RNase R? | |
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RNase R does not have a limit on the length of RNA that it can degrade. Therefore, the majority of non-circular RNA with the exception of short double-stranded RNA, 5S RNA and tRNA will be digested.
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| What inhibits RNase R? | |
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Activity of the enzyme will be abolished if EDTA is present at a concentration of 1 mM or higher. Therefore, if using EDTA compensate by adding MgCl₂ to 1.0 mM final concentration.
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| How is RNase R inactivated/removed? | |
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While the enzyme can be heat inactivated, the procedure is not recommended since high heat can lead to RNA damage. Phenol-chloroform precipitation can be used instead. For NGS, solid phase reversible immobilization (SPRI) bead cleanup is recommended.
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| Can I use higher temperatures (50°C) for my sample incubation? | |
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While some of the literature reports that RNase R enzyme is active at temperatures equal or slightly higher than 50°C, we do not recommend exceeding the suggested range of 37-45°C. For NGS applications, incubation at 37°C is recommended. This will ensure optimal activity of the enzyme and absence of non-enzymatic degradation.
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| Can RNase R be used for total RNA isolation in addition to RNA degradation? | |
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Yes, RNase R can be used for both RNA degradation and total RNA isolation. It efficiently degrades linear RNA, leaving circular RNA and genomic DNA intact.
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| Can RNase R be used for RNA integrity assessment in RNA-seq experiments? | |
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Yes, RNase R is commonly used for assessing RNA integrity before RNA-seq experiments. It helps confirm the presence of intact RNA and can be a valuable quality control step.
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| Can RNase R be used for RNA samples in difficult matrices, such as tissues or formalin-fixed paraffin-embedded (FFPE) samples? | |
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RNase R can be used for a variety of sample types, including tissues and FFPE samples. However, sample preparation methods should be optimized for challenging samples (such as incubation time and reactions conditions).
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| Is RNase R suitable for all RNA types, including eukaryotic and prokaryotic RNA? | |
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RNase R is suitable for degrading both eukaryotic and prokaryotic RNA. It is a broad-spectrum ribonuclease.
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| Does RNase R require special precautions for handling and disposal? | |
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RNase R is not hazardous. However, it is important to use appropriate lab safety practices and dispose of waste materials according to local regulations.
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| Is there any risk of contamination of RNase R with DNase or other contaminants? | |
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RNase R is rigorously tested for purity and is free from contaminants, including DNase. It is provided in a ready-to-use format.
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| Why should I choose RNaseOFF over other ribonuclease inhibitors? | |
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RNaseOFF offers enhanced resistance to oxidation, making it more stable than traditional human RNase inhibitors. It is also stable at low concentrations of DTT (< 1 mM), ensuring reliable RNA protection in a variety of experimental conditions.
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| Is RNaseOFF suitable for PCR, RT-PCR, RNA isolation, and other RNA-based applications? | |
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Yes, RNaseOFF is ideal for PCR, RT-PCR, RNA isolation, cDNA synthesis, qPCR, and other RNA-based applications. It effectively prevents RNA degradation without inhibiting polymerase activity, ensuring RNA integrity and high-quality results in all these experiments.
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