Biology Reference
In-Depth Information
B. RNA Isolation Method
Add
100
m
L of Trizol (Invitrogen) to the frozen worm pellet for a total volume
of 200
m
L. Grind worms in Trizol with a hand-held electric homogenizer that fits
1.5 mL tubes until the solution is cloudy and no clumps are visible. Add 800
m
Lof
Trizol to lysed sample, mix, and let sit at room temperature (RT) for at least 5 min.
After all samples have been homogenized as above, add 200
m
L of chloroform:
isoamyl alcohol (24:1) to each sample, shake for 30 s, and let sit at RT for 3 min. Spin
samples at 12,000g for 15 min at 4
C. Transfer the RNA-containing supernatant to
an equal volume of chloroform:isoamyl alcohol. Shake to mix, and spin at top speed
for 5 min at RT. Transfer the supernatant to an equal volume of isopropanol and 1
m
L
of 20 mg/mL glycogen. Mix samples briefly and incubate at
20
C for at least 1 h,
which is important for efficiently precipitating small RNAs. Spin samples at
12,000 g for 10 min at 4
C. Carefully remove the supernatant and add 1 mL of
75% ethanol to the RNA pellet. Spin samples at 12,000 g for 5 min at 4
C. Carefully
remove all of the supernatant. Briefly dry pellets in a 50-58
C oven for
5 min or at
RT for
10 min. Resuspend pellets in 200
m
L of DEPC water preheated to
68
C.
It is very important to thoroughly resuspend the RNA at this step and ensure that the
pellet has gone into solution entirely.
Next, a second RNA-extraction step is performed to further purify the RNA. If
samples are to be analyzed by RT-PCR, a DNase treatment can be performed as
described below in Section II before continuing with the second RNA extraction.
Add 20
m
L of 3 M NaOAc pH 5.2 and 220
m
L of phenol:chloroform:isoamyl alcohol
(25:24:1) to each RNA sample. Vortex briefly tomix, and spin at top speed for 5 min at
RT. Transfer supernatant to 220
m
L of choloroform:isoamyl alcohol (24:1). Vortex
briefly and spin at top speed for 5 min at RT. Transfer supernatant to 220
m
Lof
isopropanol and 1
m
L of 20 mg/mL glycogen. Vortex samples briefly and precipitate
by incubating at
20
C for at least 1 h. Spin samples at 12,000 g for 10 min at 4
C.
Carefully remove the supernatant and add 1 mL of 75% ethanol to the pellet. Spin
samples at 12,000 g for 5 min at 4
C. Carefully remove all of the supernatant. Briefly,
dry pellets in a 50-58
Covenfor
5 min or at RT for
10min.Dependingonthe
pellet size, resuspend pellets in 30-50
m
L of DEPC water preheated to
68
C.
Determine RNA concentration by diluting RNA 100-fold in 1
Tris EDTA (TE)
buffer pH 7.4 and analyzing the A260 value by spectrophotometry. Pure RNA typi-
cally has an A260/A280 reading of 1.8-2. An A260/A230 ratio less than 2 or 1.5
signifies contamination by genomic DNA or extraction chemicals, respectively.
II. Analysis of Primary, Precursor, and Mature miRNA Expression
Mature 22 nt miRNAs originate from genome-encoded primary miRNA (pri-
miRNA) transcripts that are mostly transcribed by RNA polymerase II, capped,
polyadenylated, and multiple kilobases in length (
Davis and Hata, 2009
).
Cleavage of the pri-miRNA in the nucleus by the RNase III enzyme Drosha in
association with Pasha produces the
70 nt precursor miRNA (pre-miRNA) hairpin
(
Davis and Hata, 2009
). Further cleavage by the RNase III enzyme Dicer in the
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