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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.

@2004 Cebra-Thomas

Last Modified: 17 August 2004

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