Please find the following protocol:

3' Rapid Amplification of cDNA Ends (RACE) by PCR



Overview:

This protocol describes 3'-end amplification of mRNA by PCR. The first-strand cDNA is synthesized from total or poly(A+) RNA by priming from the poly A tail of the mRNA using a oligo (dT) adaptor primer. The cDNA is then amplified via PCR using a gene-specific primer and an adaptor primer.



Procedure:


Synthesis of First Strands of cDNAs Using a (dT)-Adaptor Primer

1. Dissolve 1 to 5 μg of poly (A)+ RNA or 10 to 20 μg of total RNA in 10 μl of DEPC-treated water. To minimize degradation of RNA by RNases, wear gloves when handling samples and reagents, and change gloves regularly while working. Treat water and solutions with DEPC (diethyl pyrocarbonate) to inactivate RNases, and use solutions prepared with RNase-free water and equipment.

2. Incubate the RNA at 65°C for 5 min.

3. Chill the RNA on ice.

4. Add the following to the RNA:
2 μl of 10X PCR Buffer
2 μl of 10 mM dNTPs
2 μl of 10 mM DTT
0.5 μl of RNasin
3 μl of 100 ng/ml (dT)17-Adaptor
1 μl of AMV Reverse Transcriptase*

*AMV Reverse Transcriptase is used because it is more stable at higher temperatures than other reverse transcriptases. This allows for the reaction to proceed at 55°C, if so desired, to eliminate potentially inhibitory RNA secondary structures.

5. Incubate the reaction at 42°C for 1 to 2 hr.

6. Incubate the reaction at 95°C for 5 min to stop the reaction.

7. Dilute the reaction mixture with 180 μl of TE (cDNA pool). There is no need to purify the cDNA because the RNase H activity of the AMV Reverse Transcriptase degrades the RNA template.

Amplification of the Target cDNA

1. Use 1 to 10 μl aliquots from the above cDNA pool and add the following:


3. Combine 10 μl aliquots of the PCR products with 5 μl aliquots of Sucrose Loading Dye.

4. Load the samples on an Agarose Gel for analysis of the reaction products. Southern Blot analysis can also be performed using an internal gene-specific probe.


Solutions:


DEPC-ddH2O
0.1% (v/v) Diethyl Pyrocarbonate (DEPC) in ddH2O
Shake or stir the solution
Autoclave for 15 min to inactivate the DEPC
(CAUTION Biohazard!)
Allow solution to sit for at least 12 hr

Adaptor
50 ng/μl Adaptor
5'-GAC TCG AGT CGA CAT CG-3'

10 mM dNTPs
10 mM dATP
10 mM dTTP
10 mM dGTP
10 mM dCTP

Gene-specific Primer
50 ng/μl anti-sense primer to gene of interest

10X PCR Buffer
15 mM MgCl2
500 mM KCl
100 mM Tris-Cl, pH 8.3

(dT)17-Adaptor
5'-GAC TCG AGT CGA CAT CGA TTT TTT TTT TTT TTT TT-3'
100 ng/μl (dT)17-Adaptor

Sucrose Loading Dye (6X)
0.25% (w/v) Xylene Cyanol
0.25% (w/v) Bromophenol Blue
40% (w/v) Sucrose

2 mM dNTPs
2 mM dGTP
2 mM dCTP
2 mM dATP
2 mM dTTP
10 mM DTT



Bioreagents and Chemicals:

Oligonucleotide
Bromophenol Blue
Xylene Cyanol
AMV Reverse Transcriptase
dCTP
RNasin
dTTP
dGTP
Sucrose
dATP
Magnesium Chloride
Isoamyl Alcohol
Chloroform
Phenol
Potassium Chloride
Tris
Potassium Cacodylate
Diethyl Pyrocarbonate (DEPC)
DTT
Cobalt Chloride
(dT)17-Adaptor
DNA Polymerase, Taq


Reference and Links:

1. Frohman, MA, Dush, MK, Martin, GR. Rapid production of full-length cDNAs from rare transcripts: amplification using a single gene-specific oligonucleotide primer. Proc. Natl. Acad. Sci. USA 1988; 85:8998-9002.

http://www.bio.com/protocolstools/protocol.jhtml?id=p1243