| Products/Services Used | Details | Operation |
|---|---|---|
| Gene Synthesis | For in vitro assays, modSpinach, FASTmiR122 and FASTmiR171 constructs were synthesized with GenScript (Piscataway, NJ, USA) (vector pUC57-Kan). The positive control and the small RNA sensorsequencesweresynthesizedwithGenScript(Piscataway, NJ, USA) with the att sites corresponding with Gateway vector pGWB461 (P35S-TagRFP-attR1-Cmr-ccdB-attR2TNOS).For expression of Spinach and FASTmiR in mammalian cells, the positive control and the small RNA sensor sequences were synthesized with GenScript, and each piece was amplified with BamHI and XbaI sites using primers 9 and 10 (for the original Spinach), 11 and 12 (for the modified Spinach), 13 and 14 (for FASTmiR122), 15 and 16 (for FASTmiR171),21and22(forSpinach2),withallprimersequenceslistedinTableS1. | Get A Quote |
Small RNAs， including microRNAs (miRNAs) and small interfering RNAs (siRNAs)， play a variety of important regulatory roles in many eukaryotes. Their small size has made it challenging to study them directly in live cells. Here we describe an RNA-based fluorescent sensor for small RNA detection both in vitro and in vivo， adaptable for any small RNA. It utilizes an sxRNA switch for detection of miRNA-mRNA interactions combined with a fluorophore-binding sequence 'Spinach'， a GFP-like RNA aptamer for which the RNA-fluorophore complex exhibits strong and consistent fluorescence under an excitation wavelength. Two example sensors， FASTmiR171 and FASTmiR122， can rapidly detect and quantify the levels of m... More
