Molecular Techniques and Methods

Direct Cloning by mRNA Differential Display

Copy Right © 2001/ Institute of Molecular Development LLC

INTRODUCTION

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.




    PROCEDURES

    Isolation of RNA

    1. Prepare total RNA or mRNA treated with DNase (RNase-free).


    Single-Strand cDNA Synthesis

    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.

    4. Add 16.5 ul of the Reverse Transcription Mix.

    5. Incubate for 2 minutes at room temperature.

    6. Add 1.5 ul (300 units) of SuperScript RNase H-minus reverse transcriptase.

    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.

    16. Load 2 ul onto a prerun 6% polyacrylamide gel without urea and run in TTE or TBE buffer at 50 watts.

    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.

    26. Pick six white colonies from each cloned fragment and make plasmid minipreparations.

    27. Cut plasmids with restriction enzymes and check for inserts by agarose gel electrophoresis.

    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

  • Ito T, K Kito, N Adati, Y Misui, H Hagiwara, Y Sakaki (1994) Fluorescent differential display: Arbitrarily primed RT-PCR fingerprinting on an automated DNA sequencer. FEBS Lett. 551: 231- 236.

  • Liang P, AB Pardee (1992) Differential display of eukaryotic messenger RNA by means of the polymerase chain reaction. Science 257: 067-971.

  • Liang P, L Averboukh, AB Pardee (1994) Method of differential display. Methods Mol. Genet. 5: 3-16.

  • Liang P, W Zhu, X Zhang, Z Guo, RP O'Connell, L Averboukh, F Wang, AB Pardee (1995) Differential display using one-base anchored oligo-dT primers. Nucleic Acids Res. 22: 5763-5764.

  • Mou L, H Miller, J Li, E Wang, L Chalifour (1994) Improvements to the differential display method for gene analysis. Biochem. Biophys. Res. Commun. 199: 564-560.

  • Sokolov BP, DJ Prockop (1994) A rapid and simple PCR-based method for isolation of cDNAs from differentially expressed genes. Nucleic Acids Res. 22:4000-4015.

  • Welsh J, M McClelland (1990) Fingerprinting genomes using PCR with arbitrary primers. Nucleic Acids Res. 18: 7213-7218.

  • Welsh J, K Chada, SS Dalal, R Cheng, D Ralph, M McClelland (1992) Arbitrarily primed PCR fingerprinting of RNA. Nucleic Acids Res. 20: 4965- 4070.

  • Williams JKG, AR Kubelik, KJ Livak, JA Rafalski, SV Tingey (1990) DNA polymorphisms amplified by arbitrary primers are useful as genetic markers. Nucleic Acids Res. 18: 6531- 6535.


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