Biomedical Engineering Reference
In-Depth Information
Before starting an RT or PCR procedure, clean off your benchtop and pipetters
throughly (solutions such as RNase-ZAP from Ambion can be helpful). During
all procedures, use a different pipet tip each time you draw off any enzyme or
RNA, to avoid cross-contamination. Because PCR is so sensitive, if practical it is
best to keep the products of PCR reactions in a different refridgerator or freezer
from PCR primers, buffers, and so forth.
3. When working with rodent brains, following decapitation I rapidly dissect out
striatum on an ice-cold surface (e.g., roughened, up-ended culture plate in an ice
bucket), then immediately place the tissue into a prechilled microcentrifuge tube
sitting in dry ice. When all samples were collected they are placed in a -80°C
freezer. RNA extraction (which can be weeks or even months later) involves rapid
homogenization in a guanidine thiocyanate based solution, such as TriReagent
(Molecular Research Center, Inc.). It can be helpful to include a coprecipitant
(e.g., Microcarrier TR, Molecular Research), especially if working with a smaller
amount of RNA. Follow the manufacturer's protocol for RNA isolation, redissolve
RNA in DEPC-treated H
2
O (e.g., 50
L for one rat striatum's worth of RNA). Do
not
allow the RNA pellet to dry completely before adding DEPC-treated water
or it will be very diffi cult to dissolve; incubation in a warm (58°C) shaking
incubator (thermomixer) can help. Place in a 37°C bath or incubator and remove
any DNA by adding to each 50-
µ
µ
L sample the following: 0.5
µ
L of RNasin,
5.7
L of DNase I. Incubate for 1 h, then remove
the enzyme followed by phenol-chloroform (3
µ
L of 10X DNAse buffer, and 1
µ
1), chloroform extractions (the
Phase-Lock Gel I tubes provided by FivePrime-ThreePrime Inc. are useful for
avoiding waste and contamination during these extractions). Precipitate out RNA
by adding 5
L of ethanol and placing on
dry ice for 30-60 min followed by cold centrifugation at high speed (12,000
g
or more, 15 min). Carefully remove supernatant, wash with 70% ethanol, allow
pellet to dry
briefl y
(until all visible liquid is gone; this time, redissolve RNA
in a smaller volume (10-20
µ
L of 3
M
sodium acetate and 250
µ
L for a rat striatum), then carefully measure RNA
concentration in duplicate with a spectrophotometer. The A
260
/A
280
ratio of
the RNA should be approx 1.8 or higher. Avoid repeated freeze-thawing of your
RNA samples, so if the same samples will be used many times, adjust each
of the samples to the same concentration (preferably 1
µ
L or higher) with
DEPC-treated water and split into aliquots before freezing at -80°C.
µ
g/
µ
4. In some experimental designs a test can be performed at this stage to ensure that
the animal(s) exhibited the desired genetic response. For example, if it is already
known that gene X is induced by a drug at a particular timepoint, and the object
of the DDPCR experiment is to fi nd other genes that respond in a similar fashion,
the extracted RNA could be used for a Northern blot to ensure that gene X is in
fact being induced in these particular samples. Although this creates extra work,
RNA from a given rat striatum can be used for months of DDPCR gels, so it is
often worth confi rming that you are not wasting your time.
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