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AAT Bioquest

RNA Detection

Detecting and quantitating small amounts of RNA is extremely important for a wide variety of molecular biology procedures such as measuring yields of in vitro transcribed RNA and measuring RNA concentrations before performing Northern blot analysis, S1 nuclease assays, RNase protection assays, cDNA library preparation, reverse transcription PCR, and differential display PCR. The most commonly used technique for measuring nucleic acid concentration is the determination of absorbance at 260 nm. The major disadvantage of the absorbance-based method is the interferences caused by proteins and other UV absorbing compounds.

RNA dose responses were measured with StrandBrite™ Green Fluorimetric RNA Quantitation Kit
RNA dose responses were measured with StrandBrite™ Green Fluorimetric RNA Quantitation Kit (Cat# 17655) in a solid black 96-well microplate.
The major challenge to analyze RNA in live cells is the interferences caused by DNA. To address the difficulties, AAT Bioquest has developed the StrandBrite™ RNA Green, an excellent RNA-selective probe that generates significantly enhanced green fluorescence upon binding to RNA. StrandBrite™ RNA Green readily gets into live cells. It has the excitation/emission of 490/540 nm. In the DNase digest test, no significant change of fluorescence intensity in fixed cells stained with StrandBrite™ RNA Green was observed. In contrast, after RNase digestion, the initial fluorescence signal decreased immediately. These results indicate that initial fluorescence signal was generated from the specific interaction of StrandBrite™ RNA Green with RNA in cells. Short exposure of live cells to antinomycin D did cause inhibition of RNA synthesis during 6 hours after drug removal in a dose-dependent manner. This data demonstrates that StrandBrite™ RNA Green is a sensitive RNA-selective dye for staining nucleolar RNA in live and fixed cells. StrandBrite™ RNA Green has less DNA interferences than the commonly used SYTO® RNASelect™ dye. Due to its excellent cell permeability and spectral properties, it has been successfully used for flow cytometric RNA analysis and fluorescence microscope in live cells.
 
RNA dose response measured with StrandBrite™ Green Fluorimetric RNA Quantitation Kit
RNA dose response measured with StrandBrite™ Green Fluorimetric RNA Quantitation Kit (Cat#17657) in a solid black 96-well microplate using a Gemini microplate reader (Molecular Devices).
StrandBrite™ Green RNA Quantifying Reagent (Cat# 17610) is an ultrasensitive fluorescent nucleic acid stain for quantitating RNA in solution. StrandBrite™ Green RNA Quantifying Reagent can detect as low as 5 ng/mL RNA with a fluorescence microplate reader or fluorometer. StrandBrite™ Green Fluorimetric RNA Quantitation Kits (Cat# 17655, 17656 & 17657) include our StrandBrite™ RNA Green nucleic acid stain with optimized and robust protocols. They provide convenient methods for quantifying RNA in solutions.
 
Cell Navigator® Live Cell RNA Imaging Kit (Cat# 22630) includes StrandBrite™ RNA Green as it specifically binds RNA in cells. Compared with commercial SYTO® RNASelect™ dye for RNA staining in vivo, StrandBrite™ RNA Green shows a brighter signal and much better selectivity to RNA. In addition, this kit can stain RNA in both living cells and fixed cells.

Cell Navigator® Live Cell RNA Imaging Kit
Cells fixed in methanol
After staining

Fluorescence images of RNA staining in HeLa cells. Left: Live cells were stained using Cell Navigator® Live Cell RNA Imaging Kit (Green, Cat# 22630) and counter-stained with Hoechst 33342 (Blue, Cat# 17530). Center: Cells fixed in methanol were stained using the same kit. Right: After staining, fixed HeLa cells were incubated with 0.5 mg/mL RNase at 37 °C for 1 hour. Image of RNase digest test indicated the high selectivity of StrandBrite™ RNA Green. The green fluorescence signal was measured using a fluorescence microscope with FITC filter.

 

Table 1. RNA Detection Assays and Probes

Cat No.
Product Name
Ex (nm)
Em (nm)
Unit Size
22630 Cell Navigator® Live Cell RNA Imaging Kit *Green Fluorescence*503511100 Tests
17656 StrandBrite™ Green Fluorimetric RNA Quantitation Kit500525100 Tests
17655 StrandBrite™ Green Fluorimetric RNA Quantitation Kit *Optimized for Microplate Readers*5005251000 Tests
17657 StrandBrite™ Green Fluorimetric RNA Quantitation Kit *High Selectivity*500525100 Tests
17610 StrandBrite™ Green RNA Quantifying Reagent *200X DMSO Solution*5005251 mL
17611 StrandBrite™ Green RNA Quantifying Reagent *200X DMSO Solution*50052510 mL