mRNA differential display uses primers of arbitrary sequence to
generate cDNA tags. This method uses combinations of 10-mer arbitrary
primers with anchored cDNA primers and generates fragments that
originate mostly from the poly(A) tail and extend about 50-600
nucleotides upstream. mRNA differential display is able to detect
differences in gene expression of a certain percentage of expressed
genes.
The strategy of the method consists of five steps.
(1) Reverse transcription in fractions using a set of anchored primers.
(2) PCR amplification of cDNA species from each fraction using a set of arbitrary primers
and anchored primers.
(3) Electrophoretic separation of the resulting fragments.
(4) Reamplification of fragments that are different between two situations, cloning,
and sequencing.
(5) Confirmation of differential expression by an independent RNA analysis technique
(Northern blotting, RNase protection, or nuclear run-on analysis).
(1) The method uses either total RNA or mRNA as the starting material;
it is first reverse-transcribed to yield single-stranded cDNA. A clever subdivision of the total
number of mRNAs is required to be able to display as many different
expressed genes as possible. Subdivision of the mRNAs should provide
an anchor sequence for the subsequent PCR amplification. Liang
and Pardee (1992) originally suggested using 12 different primers
of the type dT12MN, where M can be A, G, or C, and N can be any of the four nucleotides.
By using these primers, one could generate 12 subfractions of
cDNA, which should represent almost equally 1/12 of the expressed
genes of a particular cell. Assuming 15,000 genes to be expressed
in a cell at one time, one subfraction of cDNA would contain cDNA
species representing 1,250 different genes.
(2) The next step is the PCR amplification of as many of the cDNA species as possible in a PCR reaction.
The anchored primers used for reverse transcription serve as the
downstream primers for this step. The upstream primers are 10-mer
oligonucleotides of arbitrary sequence; not every 10-mer is suitable
and must be tested experimentally; the lack of self-complementarity
and the same GC content (50%) of all primers are important factors.
From theoretical calculations, the chance for every mRNA species
to be displayed requires that 24-26 different primers must be
used in combination with every downstream primer, amounting to
288 or 312 individual PCR procedures. Optimal conditions for annealing
of the primers are required to obtain the maximal number of fragments
displayed in the following electrophoretic separation.
(3) Electrophoresis can be done on sequencing gels, on nondenaturing polyacrylamide gels, or even on agarose gels. We recommend the use of nondenaturing
polyacrylamide gels because they reduce the artificial complexity
of the patterns often observed with sequencing gels due to strand
separation and incomplete addition of a terminal nucleotide by
certain thermostable DNA polymerases; they also make processing
of the bands much easier. Because radioactive nucleotides are
the preferred labeled substrates, gels are first exposed to autoradiography
with X-ray film. The bands of interest are cut out from the gel
and reamplified, and the fragments are cloned into a suitable
vector. Because of the possibility of more than one cDNA species
within one band, isolation of several colonies that should be
further characterized is advisable.
(4) Characterization of the cloned fragments should be done in two ways. First of
all, a fragment of interest should be analyzed to determine whether
it corresponds to a known or an unknown gene. This requires sequencing
of the clone(s). However, one also has to make sure that the fragment
is indeed differentially regulated and not just an artifact. This
requires using another method for detecting differences in gene
expression. Northern blot analysis or RNase protection assays are the most obvious choice. Disadvantages of these methods include
not only their labor-intensive and time-consuming performance,
but also the requirement for labeling multiple probes corresponding
to the differentially displayed cloned candidates. Nuclear run-on assays is the alternative method, which require only one labeling
reaction for every cell type studied and can incorporate an almost
unlimited number of samples.
mRNA differential display is the most flexible and comprehensive
method available for the detection of almost all genes expressed
in a particular cell and for the identification of differences
in gene expression between different cell types. This technique
has at least four advantages over differential hybridization or
differential libraries.
(a) it allows simultaneous display of all differences,
(b) it detects up-regulation and down-regulation of genes at the
same time,
(c) it allows comparison of more than two situations, and
(d) it is faster.
MATERIALS AND SOLUTIONS
T12MN Downstream Primers M can be A, G, or C, and N can be any of the four nucleotides.
Reverse Transcription Mix
5 x First-Strand cDNA Synthesis Buffer ------ 150 ul
0.1 M DTT ----------------------------------- 75 ul
100 uM dNTP mix --------------------------- 150 ul
40 units/ul RNasin ---------------------------- 19 ul DEPC-treated H2O -------------------------- 19 ul
Upstream Arbitrary 10-mer Primers
Not every 10-mer is suitable
and must be tested experimentally.
The lack of self-complementarity
and the same GC content (50%) of all primers are important factors.
RT-PCR Master Mix
10 X PCR Buffer ------------------------------ 234 ul
25 mM MgCl2 -------------------------------- 140.4 ul
[33P]dATP ------------------------------------ 11.7 ul
100 uM dNTP mix ---------------------------- 46.8 ul Taq DNA polymerase (5 U/ul) ----------------- 22 ul DEPC-treated H2O ---------------------------- 949.1 ul
10 X PCR Buffer (10 ml)
500 mM KCl ----------------------------------- 5 ml of 1 M KCl
100 mM Tris-HCl (pH 9.0) --------------------- 1 ml of 1 M Tris-HCl
0.1% Gelatin ------------------------------------ 1 ml of 1% Gelatin
1% Triton X-100 -------------------------------- 0.2 ml of 50% Triton X-100
Distilled H2O ------------------------------------ 2.8 ml
20 X TTE (1 liter)
Tris base ----------------------------------------- 215 g
Taurine ------------------------------------------- 71.3 g 0.5 M EDTA ------------------------------------- 20 ml
Add distilled H2O to make a final volume of ----- 1 liter
6% Acrylamide Gel Solution 40% Acrylamide:bisacrylamide solution (19:1) ------- 14.5 ml
20 X TTE ----------------------------------------- 5.0 ml
Distilled H2O -------------------------------------- 80.5 ml
Filter through a 0.2 um filter.
Per 50 ml of the 6% Acrylamide Gel Solution,
add 40 ul of N,N,N',N'-tetramethylenediamine (TEMED) and 200 ul
of 10% Ammonium Persulfate.
2. For cDNA synthesis, prepare reaction tubes as follows.
25 uM dT12MN Downstream Primer ------------ 3 ul
Total RNA -------------------------------------- 3.0 ul (100-300
ng)
DEPC-treated H2O ----------------------------- 7.5 ul
3. Heat for 10 minutes at 70oC and place on ice immediately.
7. Incubate for 8 minutes at room temperature followed by 1 hour
at 37oC.
8. Heat for 5 minutes to 95oC, place on ice immediately, and store at -70oC.
RT-PCR and Gel Electrophoresis of DNA Fragments
9. Prepare the following premix in a microfuge tube.
RT-PCR Master mix ------------------------- 108 ul
25 uM dT12MN Downstream Primer --------- 18 ul
cDNA from step 9 ---------------------------- 9 ul
10. Distribute 15 ul of the premixed components from step 9 into
tubes.
11. Add 5 ul (2 uM) of the Upstream Arbitrary 10-mer Primer to
each tube.
12. Run PCR as follows.
Cycles
Temperature
Time
40 cycles
95oC
30 sec
40oC
60 sec
72oC
60 sec
1 cycle
72oC
7 min
13. Background smear can be avoided if this lower stringency is
used only for the first 1-5 cycles and temperature is then raised
to 45oC for the remaining 35-39 cycles.
14. Prepare the gels during the PCR reaction.
15. Concentrate half of the RT-PCR incubation mixture (10 ul)
by vacuum and heat (10 minutes) to 2 ul. Adjust with glycerol
to 5%, xylene cyanol FF, and bromphenol blue.
17. Stop the run when the bromphenol blue runs out of the gel.
18. Dry the gel on filter paper and expose to X-ray film overnight.
Reamplification, Cloning, and Sequencing of DNA Fragments
19. Cut the bands of interest e.g., those differing between the
patterns from control cells and the test cells, from the filter
paper and transfer to microfuge tubes.
20. Add 100 ul of DEPC-treated H2O and boil for 10 minutes.
21. Take out and discard the filter paper.
22. Reamplify the eluted DNA as follows.
Eluted DNA Solution ------------------------------ 26.5 ul
10 X PCR buffer ---------------------------------- 5 ul
25 mM MgCl2 ------------------------------------ 3 ul
500 uM dNTP mix -------------------------------- 5 ul
2 uM dT12MN Downstream Primer --------------- 5 ul
2 uM Upstream Arbitrary 10-mer Primer --------- 5 ul Taq DNA polymerase (5 units/ ul) ----------------- 0.5 ul
Both primers should be the same as those used to generate the
original PCR product.
23. Do PCR reaction as follows.
Cycles
Temperature
Time
40 cycles
95oC
30 sec
40oC
60 sec
72oC
60 sec
1 cycle
72oC
7 min
24. Run 10 ul of the reaction mixture on a 2% agarose gel to check the size.
25. Clone fragments of interest into the TA vector.
28. To confirm differential regulation of individual candidate
bands, do nuclear run-on assays instead of Northern blot hybridizations. Because vector sequences can create considerable nonspecific
signals, we prefer to probe the cloned fragments.
NOTES
It must be stressed that mRNA differential display is not a totally
quantitative method so far. Even drastic differences between two
situations (e.g., a strong band from one cell type and the absence
of this band in the control cell) can occur when the actual difference
in expression levels is only five-fold and, sometimes, slightly
different intensities in a particular band position are related
to dramatic differences in gene expression. The latter can occur
when a constitutively expressed RNA fragment runs in the same
gel position as one that corresponds to a regulated gene. The
major problem is the wide spectrum of abundance of individual
mRNAs. Under the standard conditions, probably only the less abundant
class of RNA is amplified in the linear range. If the number of
cycles is reduced to 30, only a very few bands are detectable.
Probably one would have to run at least three sets of analyses
(30, 35, and 40 cycles) or three different concentrations of cDNA
to compensate for this problem.
KIT INFORMATION
REFERENCES
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