Differential Display of RNAs
The differential display protocol described here is based on the principle described by:
Liang, P. and A.B. Pardee : Differential display of eukaryotic messenger RNA by means
of the polymerase chain reaction, Science 257, 967-971 (1992)
The oligonucleotides we use are kits, contaning 20 decamers, from Operon Technologies
Inc..
I. DNase I treatment of RNA
* mix and incubate for 30 min. at 37 °C: 50 µgRNA, 10 µl RNase inhibitor (1 U/µl), 1
µl RNase-free DNaseI (10 U/µl), 5 µl 0.1 M Tris-Cl pH 8.3, 5 µl 0.5 M KCl, 5 µl 15 mM
MgCl2
* phenol extract once
* precipitate with 5 µl NaAcetate and 200 µl 100 % Ethanol
* incubate at least for 30 min at -80 °C
* spin, wash and redissolve in 20 µl 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 µg/µl with H2ODEPC, keep on ice
* set up cDNA synthesis reaction for each degenerate anchored oligo(dT) primer set:
* 4 µl 5 x reverse transcriptase buffer, 2 µl DTT (0.1 M), 1.6 µl 4dNTP mix (250 µM),
200 ng RNA, 2 µl T12MN primer (10 pmol/µl), with H2O to 19 µl
* incubate for 5 min at 65 °C and for 10 min at 37 °C
* add 1 µl MMLV reverse transcriptase (200 U/µl)
* 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 µl into each tube and then add the arbitrary primer (= decamer)
* mix for each 20 µl PCR reaction: 9.2 µl H2O, 2 µl 10 x PCR reaction buffer, 1.6 µl
4dNTP mix (25 µM), 2 µl T12MN primer, 2 µl cDNA, 0.2 µl Taq DNA polymerase (5
U/µl), 1 µl [a-33P]dCTP
* aliquot and add 2 µl arbitrary primer (2 pmol/µl)
* 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 µl of the PCR reaction with 2 µl 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 µl 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 µl 3 M NaAcetate, 5 µl glycogen (10 mg/ml), 400 µl 100 %
Ethanol
* incubate at -70 °C for at least 30 min
* spin, wash with 85 % Ethanol and redissolve in 10 µl H2O
VI. reamplification
* reamplify in a 20 µl PCR reaction (using for each fragment the appropriate T12MN arbitrary primer combination)
* mix 4 µl of the isolated DNA, 2 µl 10 x PCR reaction buffer, 1.6 µl 4dNTP mix (250
pmol/µl), 2 µl T12MN primer, 2 µl arbitrary primer (decamer), 0.2 µl Taq DNA
polymerase (5 U/µl)
* 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 µl 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