Components and Levels Tested with RNaseAlert Technology
The following tables contain the compounds tested and indicate the compatibility of the compound tested with RNase A activity.
Experimental procedure: 45 µl of each listed reagent was added to an RNaseAlert tube containing lyophilized substrate plus 5 µl of 10X RNaseAlert Reaction Buffer. These mixtures were incubated for 1 hr at 37°C and then inspected for fluorescence on a UV transilluminator. Sample fluorescence was marked "-" in the "Before Addition of RNase A" if there was no visible glow and "+" if there was visible green glow.
After UV inspection and scoring, the "-" samples were further tested by adding 100 pg of RNase A and incubating an additional hour at 37°C. These tubes were then scored as above and results listed in the "After Addition of RNase A" column. RNase A was not added to enzyme solutions possessing RNase activity.
Reagents that are compatible with the RNaseAlert technology are the ones marked "-" in the "Before Addition of RNase A" column and "+" in the "After Addition of RNase A" column.
This is not a comprehensive list of compatible reagents. It serves to illustrate some typical data using the RNaseAlert Technology. More sophisticated monitoring can be performed with the RNaseAlert QC System or RNaseAlert QC System v2 using a fluorometer. Note that tips and solid surfaces are frequently positive for RNases if gloveless hands have simply touched them.
|Ambion Kit Components Tested with the RNaseAlert Technology||Before Addition of RNase A*||After Addition of RNase A*||Comments|
|DNaseZap™ Solution 1||-||+|
|Gel Loading Buffer II||-||-||Not recommended. The dye in the loading buffer quenches RNaseAlert fluorescence.|
|MAXIscript™ Transcription Buffer, 1X||-||+|
|MEGAscript™ Reaction Buffer, 1X||-||+|
|NorthernMax™ Transfer Buffer||-||+|
|NorthernMax™-Gly Gel Loading Buffer||-||-||Inhibits RNase A activity.|
|NorthernMax™-Gly Gel Running Buffer, 1X||-||+|
|Random Decamers, 10X||-||+|
|Salmon Sperm DNA (sheared)||-||+|
|THE RNA Storage Solution||-||+|
*Reagents that are compatible with RNaseAlert technology are the ones marked "-" in the "Before Addition of RNase A" column and "+" in the "After Addition of RNase A" column.
|Chemicals and Reagents Tested with the RNaseAlert Technology||Before Addition of RNase A*||After Addition of RNase A*||Comments|
|Acrylamide/Bis-acrylamide 19:1, 4%||-||+|
|Acrylic acid||-||-||Inhibits RNase A activity.|
|Agarose Gel Loading Buffer, 10%||-||+|
|Ammonium acetate, 500 mM||-||-||Inhibits RNase A activity.|
|Ammonium persulfate, 1%||-||-||Inhibits RNase A activity.|
|ATP, 1 mM||-||+|
|Betaine, 500 mM||-||+|
|Bromophenol Blue, 0.08%||-||-||Not recommended. Bromophenol blue quenches RNaseAlert fluorescence.|
|Cesium Chloride||-||-||Inhibits RNase A activity.|
|Coommassie Destain||-||-||Inhibits RNase A activity.|
|EDTA 100-500 mM||-||+||500 mM inhibits RNase A activity.|
|Ethidium bromide||-||+||Not recommended. Ethidium bromide has orange/red fluorescence that makes reading the fluorescence of the RNaseAlert substrate difficult.|
|GTP, 7.5 mM||-||+|
|Guanidinium hydrochloride, 100-400 mM||-||+||400 mM inhibits RNase A activity.|
|Guanidinium thiocyanate, 100-400 mM||-||+||200 mM inhibits 5 pg of RNase A|
|Magnesium acetate, 200 mM||-||-||Inhibits RNase A activity.|
|Magnesium acetate, 30-100 mM||-||+|
|Magnesium chloride, 100 mM||-||+|
|MOPS Gel Running Buffer, 10X||-||+|
|NorthernMax Buffer (10X)||-||-||Inhibits RNase A activity.|
|Nuclease-free water (not DEPC-treated)||-||+|
|PEG 8000, 4%||-||+|
|Phenol||-||-||Incubation with RNase A yielded slight violet fluoresecence.|
|Potassium acetate, 100 mM, pH 8.0||-||+|
|Potassium chloride, 200 mM||-||-||Inhibits RNase A activity.|
|Potassium phosphate, 100 mM||-||-||Inhibits RNase A activity.|
|Sodium Acetate, 300 mM, pH 5.2||-||-||Inhibits RNase A activity.|
|Sodium Chloride, 10-200 mM||-||+|
|Sodium Chloride, 500 mM||-||-||Inhibits RNase A activity.|
|Sodium Deoxycholate, 0.4%||-||+|
|Sodium phosphate, 2 mM||-||+|
|TE, pH 8||-||+|
|Tris, 100 mM, pH 8.0||-||+|
|Tris, 100 mM, pH 7.0||-||+|
|Urea polyacrylamide gel solution, 6%||-||+|
|Yeast Lysis Buffer||-||+|
|Zinc acetate, 100 mM||-||-||Inhibits RNase A activity.|
*Reagents that are compatible with the RNaseAlert technology are the ones marked "-" in the "Before Addition of RNase A" column and "+" in the "After Addition of RNase A" column.
|Materials Tested with the RNaseAlert Technology||Before Addition of RNase A*||After Addition of RNase A*||Comments|
|1 week old coffee with mold||+||ND*||Glowed like the moon.|
|Agilent Bioanalyzer electrodes, touched with bare hands||+||ND||Positive.|
|Frank's bare hands **||+||ND||No RNase activity detected.|
|Gary's bare hands**||+||ND||Slight glow.|
|George's bare hands||+||ND||Glowed brightly.|
|LB with bacterial funk, contaminated||+||ND||Glowed like a Texas firefly in August.|
|Pipet tips touched with bare hands||+||ND||Positive.|
* ND = Not determined.
** 100 µl of RNase-free water was placed in the palm of the subject's bare hand. 5 µl of that water was used in the assay according to the kit protocol.
|Enzymes* Tested with the RNaseAlert Technology||Before Addition of RNase A*||After Addition of RNase A*||Comments|
|BamH1, 100 U||-||+++|
|DNase I, 10 U||-||+++|
|E. coli S30 extract, 1/10th Volume||+||ND**|
|Hind III, 100 U||-||+++|
|Klenow Fragment (Exo-) (50 U)||-||+++|
|Mung Bean Nuclease||+||ND|
|Shrimp Alkaline Phosphatase||-||+++|
|T3 RNA Polymerase / placental ribonuclease inhibitor protein mix, 100 U / 30 U||-||+++|
|T4 Polynucleotide Kinase||-||+++|
|T4 RNA Ligase||-||+++|
|T7 RNA Polymerase / placental ribonuclease inhibitor protein mix, 100 U / 30 U||-||+++|
*Tested at 10%
** ND = Not determined.