Synthesis of cDNA and PCR

For cleaner results, polyadenylated RNA can be isolated as mentioned in the libraries chapter (Chapter 37). However, large amounts of RNA are required to obtain polyadenylated mRNA, so this choice is limited by the availability of the sample. Also, the time and effort employed in the extraction of more material do not necessarily correlate with an equal increase in performance. Therefore, total RNA will be the starting material used in these protocols.

The choice of the arbitrary primer to be used for amplification is of great importance. Primers should not form secondary structures with themselves. Therefore, internal hybridization sequences and palindromes should be avoided. The length of the primer for which the protocol below is designed should be 17-20 bases. For a trial experiment, either the M13 universal or reverse primers could be used.

General considerations must be taken when the first-strand synthesis is performed. First, cDNA should be made from three different dilutions of RNA from the same sample. This is especially important, since a particular band that does not appear with the same intensity in all three dilutions has a good chance of being nonspecific. Second, at least the first time a particular RNA is used with a particular primer, a similar reaction for cDNA synthesis should be performed in the absence of reverse transcriptase. This will be an indicator of the nonspecific background created by the PCR process on a particular sample.

1. Prepare dilutions of the RNA samples at concentrations of 100, 25, and 5 ng/mL in nuclease-free water. Mix in a sterile thin-walled PCR tube (amounts are in |L):

Nuclease-free water 7.0 10X first strand buffer 2.0 0.1 M DTT 4.0

1 mM dNTPs 2.0

10 pmol/mL primer* 2.0 RNA 2.0

3. Add 5 U (1 |L) of MuTLV reverse transcriptase.

5. Heat at 94°C for 5 min to kill the room temperature. For convenience, these temperature steps can be programmed in a thermocycler. After first-strand synthesis, reactions can be stored at -20°C.

*Use arbitrary primer or oligodT.

Fig. 2. Examples of a differential display analysis. Total RNA was isolated from chicken embryos at two different embryonic stages. Five, 10, and 50 ng of total RNA were subjected to reverse transcription, and afterward to amplification for 35 cycles using the M13 reverse primer, under the conditions described in the text. The amplification products were then separated on a denaturing polyacrylamide gel (A). Several bands were selected (observe the holes punched to recover the products from the gel), and after selection, the gel was re-exposed to verify recovery (not shown). The recovered material was subjected again to PCR under the conditions described in Subheading 3.2. As markers, 50 ng of cDNA were amplified independently as described in

Fig. 2. Examples of a differential display analysis. Total RNA was isolated from chicken embryos at two different embryonic stages. Five, 10, and 50 ng of total RNA were subjected to reverse transcription, and afterward to amplification for 35 cycles using the M13 reverse primer, under the conditions described in the text. The amplification products were then separated on a denaturing polyacrylamide gel (A). Several bands were selected (observe the holes punched to recover the products from the gel), and after selection, the gel was re-exposed to verify recovery (not shown). The recovered material was subjected again to PCR under the conditions described in Subheading 3.2. As markers, 50 ng of cDNA were amplified independently as described in

6. Add to each tube 20 |L of a cocktail containing (in |L):

Nuclease-free water 14.5

10X second strand buffer 2.0 10 pmol/mL primer** 2.0

a32P dCTP (see Note 11) 1.0 Taq DNA polymerase 0.5

Subject the samples to a low-stringency amplification step: 94°C/5 min; 40°C/5 min; 72°C/5 min, followed by 30 high-stringency cycles: 94°C/1 min; 60°C/1 min; 72°C/2 min. At the end, add an additional step of 5 min at 72°C to allow the polymerase to elongate unfinished products. Transfer tubes to ice or store at -20°C if they are not going to be used immediately.

7. Transfer the reaction to a 1.5 |L microfuge tube containing 2 |L of 0.2 MEDTA, 1 |L of 10 mg/mL oyster glycogen, 43 |L of 5 M ammonium acetate, and add 260 |L of 100% ethanol. Precipitate at room temperature for at least 5 min. Spin down for 15 min at 12,000 rpm in a microcentrifuge, carefully discard the supernatant, and resuspend in 8 ||L of formamide loading dye (see Note 13).

8. Together with the samples, a labeled size marker should be also loaded. Refer to Note 14 for preparation of a size marker. Heat the samples at 100°C for 5 min, chill on ice, and load 3 |L on a 50% (w/v) urea, 1X TBE, 5% (w/v) acrylamide:bis-acrylamide (29:1), 50 cm long, 0.4-mm thick gel (see Note 15). Run at 55-60 W constant power until the xylene cyanol (the slowest migrating dye) band is about 5 cm from the bottom. In this gel percentage, xylene cyanol comigrates with DNA fragments of approx 160 bases.

9. Lift the gel with a piece of Whatman 3MM paper, and dry immediately without fixing. After drying, a little bit of baby powder might be applied to the surface of the gel to prevent sticking to the film during autoradiography. Expose the gel to a single-sided emulsioned autoradiography film (Kodak BioMax) at room temperature without intensifying screens. Be sure to place position markers, e.g., fluorescent dots, so that the gel can be perfectly aligned with the film after autoradiography.

10. A 5-h to overnight exposure should give enough signal to visualize the fingerprint pattern generated. Sometimes a difference in the footprint is not really obvious, and all lanes should be carefully checked. Bona fide products should appear in all three different dilutions (Fig. 2). If no differential bands are observed at this point, another oligonucleotide might be used (see also Subheading 3.4.).

11. Once convincing differentially expressed bands have been identified, punch holes at both sides of the band. Align the gel with the autoradiogram, and mark the position with a sharp pencil through the holes. Cut the region delimited by the

**Same arbitrary primer used for first-strand synthesis (see Note 12).

Fig. 2. (continued) (A), to check again for the appearance of the products selected. These products are now cloned, sequenced, and the major product will be considered as the "real" differentially expressed gene. Until Northern blot analysis is performed, there is no previous clue about the authenticity of the products.

pencil marks with a razor blade. and place the insert in a microfuge tube containing 100 |L of TE buffer. Close the tube firmly, heat the sample at 65°C for 1 h, and store at -20°C. After cutting the bands, the gel should be re-exposed to verify that the selected band has been accurately removed.

12. Once identity of the bands has been checked, thaw the sample, mix by vortexing, spin down the solid for 10 min, and place 5 ||L of supernatant in a tube containing (in |L):

Nuclease-free water 18.5

10X Second-strand buffer 4.0 10 pmol/mL same primer 4.0 0.1 M DTT 4.0

Taq DNA polymerase 0.5

Subject the sample to 35 cycles of amplification: 94°C/1 min; 50°C/1 min; 72°C/ 2 min. Take 5 | L at the end of the amplification, and repeat as above.

13. Load 20 ||L of each sample on a 1.2% (w/v) agarose, 1X TBE gel containing 0.2 |g/mL ethidium bromide. Bands can be seen after the first round of amplification and should definitely be seen after the second one.

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