RNA Detection

3.4 In Vitro Analysis
of the Probes

1. The fluorescence and absorption measurements are performed
   with a 0.5μM concentration of probe in 10 mm quartz cuv-
   ettes with phosphate buffer.


2. For melting analysis the absorbance at 260 nm is monitored
   during a thermal cycle (three times 25–90C in 0.5C/min)
   with 1 eq of RNA target.


3. First the fluorescence of the probe is measured in the single
   stranded state, without the target nucleic acid (seeNote 2):
   TO:λex¼485 nm,λem¼500–700 nm
   QB:λex¼560 nm,λem¼575–750 nm


4. Next, the absorption spectrum (700–220 nm, 1 nm steps) of
   the probe is measured in the same cuvette.


5. 5 eq RNA is added and the fluorescence measurement (step 3)
   is repeated for the double stranded probe.
   6.Steps 3– 5 are performed three times per analyzed probe and
   three times for the blank.


6. The average of three fluorescence measurements (corrected
   with the fluorescence of the blank and normalized by the
   absorption at 260 nm) is calculated for both the single and
   double stranded states of the probes.


7. The responsiveness of the probes is calculated with following
   formula:

I

I 0 ¼

Ids

Iss

The readout of the fluorescence intensity: TO¼535 nm,

QB¼605 nm.

9. The extinction coefficient for every wavelength is calculated as
   follows:

εðÞ¼x

AbsxεðÞ 260

AbsðÞ 260

10. The quantum yields of the single and double stranded states
    are evaluated measuring the ATTO dyes (ATTO 520 for TO
    and ATTO 590 for QB) under the same conditions as the
    probes using following formula:

QY¼

R

IProbeAbsrefQYref

R

IrefAbsprobe

In vivo mRNP Detection by FIT Probes 279