Nuclease and Protease Testing: Laboratory and Commercial Considerations
The initial quality control assay for all components is an analysis for the presence of endo- and exonucleases. This involves incubating the test component with a moderate specific activity RNA probe (1.4 x 106 cpm/µg), a supercoiled plasmid or an end-labeled, Sau3A digested plasmid. The nucleic acid and sample solution are incubated for 18 hours at 37°C, and the nucleic acid is subsequently separated on a polyacrylamide gel and visualized to check its stability. An example of an RNase detection assay is shown in Figure 1. In this assay, 50,000 cpm of a moderate specific activity 32P-labeled RNA probe was incubated with dilutions of RNase A. Such assays are extremely sensitive and are capable of detecting 0.1 pg/ml of nuclease. All of our components must pass at this level. For example, our RNase-free DNase I passes the RNase assay at a concentration of 8000 mg/ml. This is 320X the concentration typically used to degrade the plasmid template DNA after an in vitro transcription reaction. The stringency of our nuclease assays is set very high in order to assure that only nuclease-free reagents are provided in our kits.
Nuclease and protease tests are also performed with the test component at a stringency 10-1000 fold higher than that at which they are normally used. What is meant by this is that both the length of time the probe is exposed to the test solution and the concentration of the solution being tested are greater than would be used under standard conditions. For example, the MAXIscriptÃ Transcription Buffer is normally used in in vitro transcription reactions at a 1X concentration for 60 minutes. However, its basal test level for the presence of nucleases would be at a 2X concentration for 18 hours (32 fold higher than standard usage). To ensure stability of transcribed RNA generated in a MAXIscript reaction, a labeled RNA probe is synthesized but then allowed to continue incubating in the transcription reaction overnight at 37°C. Only components that retain a fully intact RNA probe after this incubation will pass Ambion's quality control. Our cloned T7 phage polymerase passes nuclease quality control standards at a level 720 fold higher than what would normally be used in an in vitro transcription reaction.
An extremely important quality control assay for any enzyme or solution that will come in contact with an enzyme is to ensure that they are protease-free. Since many of the enzymes we provide are cloned, we must ensure that they are devoid of any proteases derived from the E. coli cells in which the cloned enzyme genes were grown. This analysis involves incubating a saturating amount of the test component with resorufin-labeled casein. If proteases are present, resorufin is released and detectable spectrophotometrically. This ensures that our enzymes will remain stable during long term storage.
Another significant aspect of quality control is to ensure that the kits we release exceed our guaranteed performance levels. For instance, our T3 and T7 MEGAscriptÃ kits are guaranteed to synthesize at least 80 ug of RNA per 20 µl reaction with the control DNA template. However, we do not release kits that just meet this specification. Through extensive optimization we expect to routinely reach yields of 120 µg of RNA with the pTRI-Xef1* Control plasmid. This ensures that everyone who uses a MEGAscript kit will get an extremely high yield of exceptional quality RNA.
One of the biggest frustrations of working with RNA is the ubiquitous nature of RNases which can ruin experiments. Many potential sources of RNase contamination exists from skin to bacterial nucleases. Often, contamination can come from a pipettor or glassware that was previously used with an RNase, bacteria or a contaminated solution. One of the best methods for decontamination is to wipe down pipettors, glassware and work surfaces with Ambion's RNaseZap™ (Cat. #9780 and 9782). RNaseZap works immediately on contact and completely inactivates nucleases and proteases. Many of our customers use our nuclease protection assay kits which require pipetting both RNA and RNases. If you do not have a dedicated set of pipettors solely for working with nucleases, it is possible that you will experience RNase contamination problems at some point in time. A dedicated set of pipettors for use with RNase is a good way to prevent RNase contamination. However, if you lack this luxury, a quick treatment of your pipetterwith RNaseZap should alleviate carry-over of nucleases into precious RNA samples.
If you suspect that one of your solutions is contaminated, you can use Ambion's Quality Control Assay for RNase described below, in "Testing for RNase in the Laboratory", and shown in Figure 1. However, the extreme sensitivity of this assay (0.1 pg/ml) is usually not necessary and may pick up very minor contaminations that in all likelihood would not affect the outcome of the experiment performed with the solution. An alternative, Ambion's RNaseAlertÃ Kit (Cat. #1964), allows researchers to quickly identify contaminated solutions without having to generate an RNA probe or use radioactivity.
Our goal at Ambion is to provide the highest quality reagents and kits. We put all of our products through rigorous nuclease, stability and functional testing. Furthermore, all Ambion products are used daily in our own Research and Development laboratories to add a further level of quality control. We believe that the extreme levels of quality control that we subject our products to will provide you with high quality products for molecular biology that are second to none.
Testing for RNase in the Laboratory
- Nuclease-free H2O
- MAXIscriptÃ 10X Transcription Buffer or 10X concentration of an appropriate medium salt buffer (see note below)
- Moderate specific activity RNA (e.g. generated with the MAXIscript Kit)
Note: It is important that this buffer contain DTT and divalent cations (e.g. Mg2+) as they may be required for nuclease activity. Without these components, solutions containing contaminating nucleases may go undetected until used in experiments where DTT and divalent cations are present.
Add the following to a microfuge tube:
- 1 µl of RNA probe
- 1 µl of 10X Transcription buffer
- 1-8 µl of solution to be tested
- Nuclease-free H2O to 10 µl final volume
Negative control reaction:
- 1 µl of RNA Probe
- 1 µl of 10X Transcription buffer
- 8 µl of nuclease-free H2O
Positive control reaction:
- 1 µl of RNA Probe
- 1 µl of 10X Transcription buffer
- 1 µl of RNase A (1 pg/ml)
- 7 µl of nuclease-free H2O
Caution: Use only the designated pipettors to pipet RNase or clean pipettors afterwards with RNaseZap!
Incubate reaction at 37°C for 16 to 24 hours
- Prepare a 5% acrylamide/8 M urea gel.
- Add 10 µl of denaturing gel loading buffer (e.g. Ambion's Solution E) to each sample.
- Heat samples at 95°C for 2-3 minutes.
- Load 10 µl of each sample on the gel.
- Run the gel until the bromophenol blue just runs off the bottom of the gel.
- Expose the gel to film for 15-30 minutes and develop.
Note: If your RNA Probe in the Negative Control reaction is degraded, the entire experiment should be repeated since an external contamination event has occurred. Wipe pipettors and work area with RNaseZap!
If your RNA Probe in the Positive Control lane is not degraded, the assay was not sensitive enough and must be repeated.
The resulting autoradiograph should show a single, crisp band that is roughly of equal intensity with the signal in the Negative Control lane. To pass Ambion's RNase quality control assay, the RNA probe must be within 20% of the intensity of the Negative Control lane with no sign of degradation (downward smearing from band). If instead you see a smear/degradation or significantly less of the RNA probe, the component being tested contains nuclease contamination.
As previously indicated, this assay is extremely sensitive, but drawbacks are the use of the radioactively labeled RNA transcript, the labor involved and the turnaround time. As an alternative, Ambion has developed a nonisotopic kit for the detection of nucleases that is fast and requires minimal hands on time. This is our RNaseAlertÃ Kit. The RNaseAlert Kit uses a novel RNA substrate tagged with a fluorescent reporter molecule (fluor) on one end and a quencher on the other. In the absence of RNases, the physical proximity of the quencher dampens fluorescence from the fluor to extremely low levels. When RNases are present, however, the RNA substrate is cleaved and the fluor and quencher are spatially separated in solution. This causes the fluor to emit a bright green signal (when excited by light of the appropriate wavelength) that can be readily detected by eye (by illumination on a UV box) or with a filter-based or monochromator-based fluorometer. Since the fluorescence of the RNaseAlert Substrate increases over time when RNase activity is present, results monitored with a fluorometer can be evaluated kinetically. The sequence of the RNaseAlert Substrate has been carefully optimized to detect several RNases, including RNase A, RNase T1, RNase I, micrococcal nuclease, S1 nuclease, mung bean nuclease, and Benzonase®. The RNaseAlert technology is extremely sensitive and can detect 0.5 pg RNase A or less.