In vitro transcription and RNA labeling
Large Scale RNA Production Systems-T7 (#P1300,
Promega) was used according to manufacture’s protocol. Thaw reagents at room temperature and
mix the transcription buffer and the rNTPs by a vortex. Make sure to combine
the reaction components at room temperature to avoid precipitation of DNA or
dNTPs in the presence of spermidine when cold. Spermidine is a component of
the transcription buffer. After mixing, incubate at 37 °C for 4 h or
T7 Transcription 5X
100 mM rATP 7.5
100 mM rUTP 7.5
100 mM rGTP 7.5
100 mM rCTP 1
(3,000 Ci/mmol, 10 μCi/μl) 2
μl if fresh, otherwise if 2 half-lives has passed use 3–5 μl
Purified template DNA
(5–10 mg) in nuclease-free water 44.5
μl (5–10 μg)
Enzyme Mix (T7) 10
Removal of template DNA
procedure in the RiboMAXTM Large Scale RNA Production
- Add RQ1 RNase-Free DNase (1 U/μl) to a
concentration of 1 U/μg of the template DNA used; incubate at 37 °C
for 15 min. (e.g. when adding 5 μg of template DNA, use
10–20 μl DNase.).
It is found that this duration as recommended by the manufacturer
is not sufficient to degrade completely the template DNA used. Increase
this incubation reaction to 3–4 hours if residual DNA is detected
in the negative control used for RT-PCR (to be linked). This can cause
background amplification during SELEX and may render cycles futile
because of the persistence of initial library sequences.
- Extract with 1 volume (100 μl) of
acetate-saturated phenol (pH 4):chloroform:isoamyl alcohol mixture
Sigma). Mix by a vortex
for 1 min (mixture turns cloudy/milky), and centrifuge at 16,000 g for 2 min. Transfer the upper (aqueous) phase to a new tube or
aspirate and discard the lower phase. Take proper precaution in handling radioactive solid
and liquid waste.
- Add 1 volume (100 μl) of
chloroform:isoamyl alcohol (24:1, #C0549,
Sigma). Mix by a
vortex for 1 min and centrifuge at 16,000 g for 2 min. Transfer the upper (aqueous) layer to a new tube or
aspirate and discard the bottom phase. If residual phenol remains,
repeat centrifugation and aspirate from the bottom phase.
- Add 0.1 volume (10 μl) of 3M sodium
acetate (pH 5.2) provided and 1 volume (100 μl) of isopropanol or
2.5 volumes (250 μl) of 100% ethanol. Isopropanol is advantageous because it keeps the
unincorporated rNTPs in solution and the volume used is less than
ethanol (useful with larger volumes of phenol-extracted RNA, to be
linked to RNA elution)
- Mix by vortex and place in the freezer for
- To precipitate RNA, spin at 16,000 g or top speed for 30 min in the
temperature-controlled centrifuge at 4 °C.
- Carefully aspirate the supernate and wash
the pellet with 0.8 μl 70% ethanol. Dry the pellet at 37 °C using a heat block.
- Suspend RNA in 1 volume (100 μl) of
nuclease-free water or the riboprobe buffer that comes in Illustra
kits. Store at
−70 °C for long-term storage, or –20 °C for next-day usage,
or follow on to the next step.
Removal of unincorporated nucleotides
Use MicroSpin G-50 spin
columns (#27-5330-01, GE Healthcare)
according to the manufacturer’s procedure. Each column can be used for up to 50 μl solution to
- Pre-spin the column at 3,000 rpm (740 g) for 1 min, and collect the buffer in the
collection tube provided.
- Add the sample to be desalted onto the
centre of the G-50 resin within the tube without perturbing the resin
with the tip. Avoid
applying the sample to the sides of the tube.
- Put the column assembly into a clean
Eppendorf (O-ring capped).
This time spin for 2 min at 3,000 rpm (740 g) to collect desalted RNA.
- Store at −70 °C for long-term storage,
or –20 °C for next-day usage, or follow on to the next step.
to To-Do List