RNA Detection

9. 3% agarose/ACSF.


10. Collagen solution (e.g., Nitta Gelatin Cellmatrix).

2.5 Image and
Statistical Analysis

1. Image J (NIH).


2. Imaris (Bitplane).


3. Prism (Graphpad).

3 Methods

3.1 Designing
Effective Sequence
of ECHO Probes and In
Vitro Characterization

3.1.1 Design Probe
Sequence

Target-dependent emission efficiency of ECHO probes can be

attenuated by (1) intramolecular interaction; (2) intermolecular

self-dimerization; (3) mismatching base pairs; and (4) tertiary

structures of target RNA [21]. Additional considerations are

taken to avoid nonspecific detection by (1) sequence-homology

search against genome sequence of the target species (e.g., mm9)

to guarantee single identity of detection sequence; (2) avoid func-

tional structures of the RNA to be detected and select single-

stranded region predicted by popular RNA structure prediction

programs (e.g., M-fold, RNA-fold, Fig.1a).

1. Intramolecular and intermolecular interaction within the probe
   sequences can be predicted using NABiT software [21]; Probe
   sequences with maximal scores of 100 are usually chosen for
   synthesis.


2. Unique target identity with 100% complementarity can be
   confirmed by BLAST search against the RefSeq database.

3.1.2 Kinetic
Measurement Using
Stopped Flow Assay

Probe characterization in vitro, including kinetics analysis and

on–off ratio measurement, is used to predict how well a probe

may work in vivo.

1. Kinetic measurements are carried out at room temperature
   using a stopped-flow spectrafluorometer equipped with dual
   photomultiplier tubes. Excitation 500 nm, filter set 520/
   35 nm (Fig.1b).


2. 50 nM probes and 0.35, 0.7, 1.4, or 2.8μM target RNA/DNA
   are dissolved separately in PBS, and mixed by firing the injec-
   tion piston with simultaneous collection of fluorescence inten-
   sity as a function of time with millisecond resolution.


3. At least six experiments are performed and the average fluores-
   cence intensity value is plotted against time.


4. Activation time is the time required for the probe fluorescence
   to reach half maximal intensity (t1/2) (Fig.1b).


5. Kinetics of hybridization-dependent fluorescence activation is
   fitted toy¼ceKx+A;y: fluorescence intensity;x: lapsed time

Nuclear RNP Dynamics in Mammalian Tissue 263