Differential Display of RNAs

互联网2013-09-06

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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 MgCl 2
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 H 2O_DEPC
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 - T 12MA,T₁₂ MC, T₁₂ MG, T₁₂ MT ; where M is A,C or G)
dilute DNA-free RNA to 0.1 µg/µl with H₂ O_DEPC , 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 T₁₂ MN primer (10 pmol/µl), with H₂ O 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 PCR s 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 T₁₂ MN primer, aliquot 18 µl into each tube and then add the arbitrary primer (= decamer)
mix for each 20 µl PCR reaction: 9.2 µl H₂ O, 2 µl 10 x PCR reaction buffer, 1.6 µl 4dNTP mix (25 µM), 2 µl T₁₂ MN 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 H 2O 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 H 2 O

VI. reamplification

reamplify in a 20 µl PCR reaction (using for each fragment the appropriate T₁₂ MN - 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 T₁₂ MN 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