Differential Display PCR

Friday, June 04, 2004

Description
To identify and characterize those genes that are differentially expressed in order to understand the molecular nature of disease state and subsequently, to devise rational therapies.

Differential display of eukaryotic messenger RNA by means of the polymerase chain reaction

Procedure
I. DNase I treatment of RNA

* mix and incubate for 30 min. at 37 C: 50 gRNA, 10 l RNase inhibitor (1 U/l), 1 l RNase-free DNaseI (10 U/l), 5 l 0.1 M Tris-Cl pH 8.3, 5 l 0.5 M KCl, 5 l 15 mM MgCl2
* phenol extract once
* precipitate with 5 l NaAcetate and 200 l 100 % Ethanol
* incubate at least for 30 min at -80 C
* spin, wash and redissolve in 20 l H2ODEPC
* measure RNA concentration and check integrity on a denaturing gel

II. cDNA synthesis

* for each RNA set up four reactions (one tube for each degenerate anchored oligo(dT) primer set - T12MA,T12MC, T12MG, T12MT; where M is A,C or G)
* dilute DNA-free RNA to 0.1 g/l with H2ODEPC, keep on ice
* set up cDNA synthesis reaction for each degenerate anchored oligo(dT) primer set:
* 4 l 5 x reverse transcriptase buffer, 2 l DTT (0.1 M), 1.6 l 4dNTP mix (250 M), 200 ng RNA, 2 l T12MN primer (10 pmol/l), with H2O to 19 l
* incubate for 5 min at 65 C and for 10 min at 37 C
* add 1 l MMLV reverse transcriptase (200 U/l)
* incubate for 50 min at 37 C
* inactivate MMLV RT by incubation for 5 min at 95 C
* use immediately for PCR amplification or store at -20 C

III. PCR reaction

* all PCRs should be done in doublets and always run in the same machine (to minimize variations)
* prepare mastermix for all PCR reactions which contain the same T12MN primer, aliquot 18 l into each tube and then add the arbitrary primer (= decamer)
* mix for each 20 l PCR reaction: 9.2 l H2O, 2 l 10 x PCR reaction buffer, 1.6 l 4dNTP mix (25 M), 2 l T12MN primer, 2 l cDNA, 0.2 l Taq DNA polymerase (5 U/l), 1 l [a-33P]dCTP
* aliquot and add 2 l arbitrary primer (2 pmol/l)
* run PCR: 40 cycles (94 C, 30 sec.) (40 C, 2 min.) (72 C, 30 sec.), 1 cycle (72 C, 5 min.)
* store PCR reactions at -20 C until the gelrun

IV. denaturing gel

* prepare 6 % denaturing polyacrylamide gel (7 M urea, 1 x TBE), prerun for 30 min at 60 W
* mix 3.5 l of the PCR reaction with 2 l formamide loading buffer, incubate 3 min at 95 C, chill on ice
* load samples (together with a labelled size marker) and run gel until xylene cyanol dye nearly reaches the bottom (3 hours)
* place gel without fixation on Whatman 3MM filter paper and dry on a gel dryer
* autoradiograph for 24 to 48 hours

V. isolation of PCR fragment

* cut out band of interest with a clean razor blade
* soak in 100 l H2O for 10 min at room temperature in a microcentrifuge tube
* boil for 15 min
* spin and take supernatant (containing the DNA) into fresh tube
* precipitate with 10 l 3 M NaAcetate, 5 l glycogen (10 mg/ml), 400 l 100 % Ethanol
* incubate at -70 C for at least 30 min
* spin, wash with 85 % Ethanol and redissolve in 10 l H2O

VI. reamplification

* reamplify in a 20 l PCR reaction (using for each fragment the appropriate T12MN - arbitrary primer combination)
* mix 4 l of the isolated DNA, 2 l 10 x PCR reaction buffer, 1.6 l 4dNTP mix (250 pmol/l), 2 l T12MN primer, 2 l arbitrary primer (decamer), 0.2 l Taq DNA polymerase (5 U/l)
* use the PCR conditions as under section III.
* run PCR product on a 1.5 % agarose gel and isolate fragment of expected size
* if there is no visible PCR product, take 1 l of the first reamplification and reamplify again
* the isolated PCR fragment can be used directly for cycle sequencing, subcloning or
as probe for northern blots

Recipes
Liang, P. and A.B. Pardee : Differential display of eukaryotic messenger RNA by means of the polymerase chain reaction, Science 257, 967-971 (1992)

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Differential Display PCR