Isolation and
analysis of total RNA from chick embryos, cDNA synthesis and
PCR analysis
Objective:
to become familiar with molecular techniques used to analyze
gene expression.
Procedure:
We willisolate total RNA (a
mix of ribosomal RNA, transfer RNA and mRNA) by disrupting
the cells of the
embryo in a combination
ofphenol and quanidine
isothiocyanate (TRIZOL). This will denature all of the
cellular
proteins, including RNase.
The RNA will be separated from the remaining cellular
macromolecules by differential
extraction and precipitated
with alcohol. The RNA will be heated in the presence of
denaturing agents (formamide) to
eliminate secondary structure
and run on an agarose minigel. The 2 ribosomal RNA bands
(18S and 28S) should be
visible under UV light in the
presence of ethidium bromide. As these RNAs are very large,
this is a good indication that
the RNA sample in intact (not
degraded by RNase). Finally,
we will synthesize cDNA using a kit (Life Sciences). A
short
piece of single-stranded DNA,
oligo-d(T), will be annealed (base-paired) to the stretch of
A's present at the 3' end of most
mRNA molecules (the poly-A
tail). This will be used to prime the synthesis of a
complementary DNA strand by reverse
transcriptase. The resulting
cDNA will be stored for later analysis of gene expression by
PCR.
Note:
when working with RNA it is necessary to be
extremelyparanoid.
Wear gloves at all times. Make sure that all
solutions, tubes, pipet tips,
etc. are for RNA work. Immediately discard
anytips or tubes that may
have come in contact
with the bench or other
surface that is not RNase-free.
Isolation
of chick embryo total RNA
1. Prepare clean working area. WEAR GLOVES.
Wipe off tools with 70%
ethanol.
Prepare 3 small dishes of
Howard's
Ringers solution (DEPC-treated
to destroy RNase).
2. Wipe
of egg with 70% ethanol. Puncture egg at wide end (above air
space) using point of sharp forceps. Carefully
remove shell above air
space. Using fine forceps, peel back shell membrane to
expose embryo. Transfer embryo to
dish with Howard's. Transfer
to 2nd dish.
3. When
you and your partner(s) are finished dissecting, quickly
remove membranes surrounding embryo with fine
forceps. Immediately
before use, transfer embryos, without an excess of
liquid, to clean LABELED 8 ml tube.
4.
Wear gloves and safety
goggles. Add 1 ml TRIZOL (Sigma) and
immediatelydisrupt
embryos using polytron
homogenizer. Allow to sit at
room temperature for at least 5 minutes. Transfer 1 ml to
clean, labeled microfuge tube.
Discard tips in waste
bags.
|
