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® TechnologyBefore Addition of RNase A*After Addition of RNase A*Comments
Acetone 10%-+ 
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.
Chloroform-- 
Coomassie Destain--Inhibits RNase A activity.
DEPC-treated water-+ 
DMSO 10%-+ 
EDTA 100-500 mM-+500 mM inhibits RNase A activity.
Ethanol, 10%-+ 
Ethidium bromide-+Not recommended. Ethidium bromide has orange/red fluorescence that makes reading the fluorescence of the RNaseAlert® substrate difficult.
Glycerol-+ 
Glycerol, 10%-+ 
Glycine-+ 
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-+ 
Mineral Oil-+ 
MOPS Gel Running Buffer, 10X-+ 
NorthernMax Buffer (10X)--Inhibits RNase A activity.
NP40, 1%-+ 
Nuclease-free water (not DEPC-treated)-+ 
PBS, 0.1X-+ 
PBS, 1X-+ 
PEG 8000, 4%-+ 
Phenol--Incubation with RNase A yielded slight violet fluorescence.
Potassium acetate, 100 mM, pH 8.0-+ 
Potassium chloride, 200 mM--Inhibits RNase A activity.
Potassium phosphate, 100 mM--Inhibits RNase A activity.
RNAlater™-- 
SDS, 1%-+ 
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-+ 
TBE, 1X-+ 
TE, pH 8-+ 
Tris, 100 mM, pH 8.0-+ 
Tris, 100 mM, pH 7.0-+ 
Tween, 1.0%-+ 
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+NDPositive.
Frank's bare hands **+NDNo RNase activity detected.
Gary's bare hands**+NDSlight glow.
George's bare hands+NDGlowed brightly.
LB with bacterial funk, contaminated+NDGlowed like a Texas firefly in August.
Pipet tips touched with bare hands+NDPositive.
Saliva+NDPositive.


* 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)-+++ 
Kpn I-+++ 
Msp I-+++ 
Mung Bean Nuclease+ND 
Nco I-+++ 
Nde I-+++ 
Nuclease-free water-+++ 
Proteinase K-+++ 
RNase A+++ND 
RNase I+++ND 
RNase T1+++ND 
S1 nuclease+++ND 
Sac I-+++ 
Sal I-+++ 
Shrimp Alkaline Phosphatase-+++ 
SuperTaq-+++ 
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-+++ 
Xba I-+++ 


*Tested at 10%
** ND = Not determined.