RNA Detection

increase of the DNA intercalating dye thiazole orange (TO) [5]. TO is used as a base surrogate, replacing one of the internal ba ...

Oxidizer: 0.02 M iodine in pyridine, water, and THF 0.4:9:90.6 (v:v:v) (e.g., emp Biotech). Deblock: 6% dichloroacetic acid in ...

Oocyte cultivation medium: full S2 cell culture medium, 15% fetal bovine serum (FBS), 1% streptomycin–penicillin antibio- tics ...

2.8 Microscopy 1. Inverted confocal microscope with a low and a high magnifica- tion, high numerical aperture objective (e.g., L ...

3 Methods 3.1 Probe Design 1. Choose the target sequence (preferentially 18–20 nucleotides in length) you want to detect in cell ...

3.4 In Vitro Analysis of the Probes The fluorescence and absorption measurements are performed with a 0.5μM concentration of pr ...

The brightness is calculated as follows: Br¼ QYεðÞ 485 = 560 1000  The probes with the best fluorescence properties (brigh ...

Transfer the ovaries in a 20–25μL volume to a nontreated clean glass slide with a P200 micropipette using a cut 200μL pipette t ...

HEAT: RAMP + 5 C, PULL¼35, VEL¼75, TIME¼130 d, PRESSURE¼ 500 Insert a new capillary and execute the program. Repeatstep 3to obt ...

After the ovarioles are positioned, blot away the cultivating medium using the Whatman filter strips. This will also remove all ...

adjacent frames. The resolution of the images we typically acquire are 120 nm120 nm800 nm at 3.3–5 Hz (see Note 16). 4 Notes ...

Since PFA and formaldehyde covalently cross-link protein molecules to RNA and also break the sugar phosphate back- bone of nucl ...

the highest available pressure applied, position the stage such that the tip only slightly reaches trough the edge of the glass ...

References Tyagi S, Kramer FR (1996) Molecular beacons: probes that fluoresce upon hybridization. Nat Biotechnol 14(3):303–308. ...

Chapter 19 Visualizing RNA in Live Bacterial Cells Using Fluorophore- and Quencher-Binding Aptamers Murat Sunbul, Ankita Arora, ...

1.1 RNA Imaging Using a Fluorophore- Binding Aptamer Previously, several short RNA sequences (aptamers) which bind to specific f ...

attaching a very efficient contact quencher (DN, dinitroaniline) via a triethylene glycol linker to yield a fluorogenic dye (SR- ...

disruption of the PeT and FRET quenching upon binding of the aptamer to the quencher, the fluorescence turn-on ratios obtained i ...

PCR purification kit (e.g., Qiagen). Restriction enzymes. DNA ligase. 1% agarose and gel electrophoresis equipment. 10 mg/mL et ...

Incubator and shaker at 37C. Spectrophotometer. 2.4 Microscopy and Image Analysis 8-well Nunc™Lab-Tek™II chambered cover gla ...