Optimized Welsh DDRT-PCR Protocol

Optimized Welsh DDRT-PCR Protocol

Provided by Mirta Grifman and found in Neuroscience Protocols (Nov 1995)

MATERIALS:

Special equipment:

  1. Thermocycler for PCR, such as the Perkin-Elmer (Norwalk, CT) oil-free 9600 GeneAmp PCR system
  2. Sequencing apparatus such as Sequi-gen sequencing cells from Bio-Rad (Hercules, CA)
  3. Gel dryer (like the Bio-Rad model 583).
Chemicals and reagents (listed in order of need):

  1. RNAzolTM B from BIOTECX Laboratories (Houston,TX), MicrocarrierTM from Molecular Research Center, Inc. (Cincinnati, OH).
  2. Reverse transcriptase (RT) buffer from GIBCOBRL (Gaithersburg, MD), deoxynucleotide triphosphate mixture (dNTPs) from Pharmacia Biotech (Piscataway, NJ), dithiothreitol (DTT, GIBCOBRL), RNase inhibitor, Boehringer (Mannheim), SuperscriptII reverse transcriptase, (GIBCOBRL).
  3. Taq DNA polymerase and buffer, (Boehringer), a [32P]-dATP (3000Ci/mmol), Amersham International (Amersham).
  4. Loading buffer (90% deionized formamide, 10mM EDTA, pH 8.0, 0.025% bromophenol blue and 0.025% xylene cyanol), acrylamide, bis-acrylamide, urea (molecular biology grade), Whatmann 3MM paper, Tracker TapeTM (Amersham International), X-Ray films.
  5. Ethanol, Na Acetate.
  6. TA-Cloning Kit from Invitrogen (San Diego, CA).
  7. "Hot Tub" cycle sequencing kit (Amersham International), STET buffer (8% w/v sucrose, 0.1% w/w Triton X-100, 50mM EDTA, pH 8.0, 50mM Tris-HCl, pH 8.0), lysozyme, cetrimide (CTAB), agarose.

DETAILED PROCEDURE:

RNA preparation

Any type of RNA preparation may be used if the RNA obtained is undegraded. RNA extraction with RNAzolTM B is rapid (2 hours) and simple to perform. The expected yield is 1-1.5ug/mg brain tissue and 10ug/mg of cultured cells (aprox. 107 cells). The yield can be improved by adding MicrocarrierTM to the homogenization step. In order to overcome minor changes arising from differences between individuals, it is very important to pool tissue samples from at least three experimental animals. Special care should be taken to avoid RNA degradation, thus the homogenization step should be carried out rapidly and external RNase sources must be avoided. Other RNA extraction procedures such as the guanidinium thiocyanate-cesium chloride method2 or cytoplasmic RNA extraction4 are also recommended. Poly (A)+ mRNA purification from the total or cytoplasmic RNA can also improve the results, as amplification of mitochondrial RNA or of partially degraded transcripts is avoided. However, It was noted by some researchers that the use of mRNA can cause problems in the DD reaction, since poly dT can carry over into the PCR step.

Differential Display

I. First strand cDNA synthesis. In this step the RNA is reverse transcribed using a primer whose sequence is arbitrarily chosen. The enzyme reverse transcriptase will extend those primers which have annealed to RNA molecules to yield single stranded cDNAs. Incubate the RNA samples at 65C for 10 min and transfer immediately to ice. Reaction mixture.

Stock solutions					Final concentration
2ul 5X RT buffer				1X
1ul 100mM dNTPs					10mM
1ul 100mM DTT					10mM
1ul 10uM oligodeoxynucleotide (17-mer)*		1uM
0.25ul RNase inhibitor (40U/ul)			1U/ul
0.25ul SuperscriptII RT (200U/ul)		5U/ul
1ul RNA (0.5ug/ul)				50ng/ul
DDW (double distilled water) to 10ul
* The primer should be chosen so that its G,C content will not exceed 60%. Self complementarity must be avoided. It is advisable to test a set of primers against any new RNA source and choose those that yield the highest number of clearly distinct bands.

Place the reaction mixtures in the thermocycler. Reaction conditions: 37C, 45 min; 95C, 5 min; 4C until use.

II. Second strand cDNA synthesis and PCR amplification: Reaction mixture:

Stock solutions                  		Final concentration
5ul 10X Taq buffer				1X
5ul 10uM oligodeoxynucleotide (17-mer		1uM
0.5ul Taq DNA polymerase (5U/ul)		50U/ml
10ul cDNA (from the first strand cDNA reaction)         
0.5ul a [32P]-dATP (3000Ci/mmol)		0.1uCi/ul
DDW to 50ul
If needed overlay the reaction with two drops of mineral oil. Place the reaction mixtures in the thermocycler. Reaction conditions: Second strand cDNA synthesis: 94C, 5 min; 40C, 5 min; 72C, 5 min. (1 cycle). PCR amplification: 94C, 1 min; 55C, 1 min; 72C, 2 min. (30 cycles) and 72C, 5 min.

Reaction mixtures can be stored at -20C until the electrophoresis step, but it is suggested that this be performed no later than 72 hours after the reaction to avoid band fading.

C. Gel electrophoresis: It is advisable to run the samples together with size markers, for example a sequencing reaction, which will yield a ladder of bands of known lengths.

  1. Mix 2.5ul of each reaction mixture with 2.5ul loading buffer.
  2. Boil for 2 min, and transfer to ice.
  3. Spin down to collect the sample and load on a standard 4% polyacrylamide, 50% urea, sequencing gel.
  4. Electrophorese at 2500V until the xylene cyanol dye reaches the bottom of the gel.
  5. Dry the gel on a piece of Whatmann 3mm paper at 80C, using a gel drier.
  6. Add orientation markers with either radioactive ink or Tracker TapeTM and expose the dried gel to autoradiography (overnight should suffice for the appearance of strong bands)
  7. Develop the film.
  8. Search for differentially displayed bands among the samples. We consider a band that differs from control samples in the same manner in both duplicate reactions from 3 experiments or animals to be reliable.

Isolation and identification of differentially displayed bands.

Reamplification (should be planned for the same day as the ligation):

  1. Align the x-ray film with the dried gel adhered to Whatmann paper, cut out the bands of interest and incubate in 100ul DDW at room temperature for 10 min.
  2. Boil for 15 min to allow the DNA to diffuse out of the gel.
  3. For DNA precipitation, mix: 100ul diffused DNA 250ul ethanol 11ul 3M Na acetate, pH 5.2 2ul MicrocarrierTM or 5ul of 10mg/ml glycogen.
  4. Centrifuge for 30 min at 4C.
  5. Carefully decant supernatant .
  6. Add 500ul 75% ethanol and repeat steps (4) and (5).
  7. Dissolve in 10ul DDW.
  8. Reamplification reaction mixture:

    Stock solutions					Final concentration
5ul 10X Taq buffer				1X
1ul 40mM dNTPs (10mM each)			200uM each
5ul 10uM oligodeoxynucleotide (17-mer)		1uM
3ul DNA (from step 7)
0.5ul Taq DNA polymerase (5U/ul)		50U/ml
DDW to 50ul

If needed overlay the reaction with two drops of mineral oil.
  9. Place samples in the thermocycler. Reaction conditions: 94C, 2 min (1 cycle); 94C, 1 min; 55C, 2 min and 72C, 1 min (30 cycles) and 72C, 5 min (1 cycle). Keep at 4C until the ligation step.

Cloning:

One efficient way to clone rapidly a PCR product is to use the TA- Cloning Kit from Invitrogen. This kit takes advantage of the fact that Taq polymerases add a single deoxyadenosine at the 3' ends of the PCR products. The vector provided contains a 3' T-overhang ready for the insertion of the PCR products. Note that ligation efficiency will sharply drop upon prolonged storage or freezing of the PCR product because the A-overhand might be lost. The kit also contains competent cells, and can be used virtually as instructed. After the ligation and transformation steps, bacteria are seeded on X-gal containing agar plates, allowing for blue-white screening. Choose several (12-20) white colonies and extract DNA to verify the insertion of the PCR product in the vector.

Sequencing:

In essence, any Sanger-based technique would do. We have employed the "Hot Tub" cycle sequencing kit and have found it to be convenient for obtaining fast sequence information. The following detailed method of preparation of the template for sequencing is adapted for use with this kit. If a kit other than "Hot Tub" will be used, the template should be prepared to suit the specific kit requirements.

Template preparation:

  1. Grow a 1.5ml culture overnight at 37C (shaking) from each colony to be analyzed.
  2. Spin the cells and discard supernatant.
  3. Dissolve the pellet in 200ul STET buffer.
  4. Add 4ul of 50 mg/ml lysozyme.
  5. Boil for 45 sec and centrifuge for 10 min at room temperature.
  6. Lift out the pellet with a toothpick and discard it.
  7. Add 8ul 5% CTAB and centrifuge for 5 min.
  8. Discard the supernatant and dissolve pellet in 300ul of 1.2M NaCl.
  9. Add 750 ul cold ethanol and centrifuge for 10 min at room temperature.
  10. Wash the pellet with 75% ethanol. Dry the pellet and dissolve in 20ul DDW.
  11. Electrophorese in a 1% agarose gel containing ethidium bromide to verify the template concentration.
For the cycle sequencing reaction follow the kit instructions. It is suggested to use a 100-500 fmol template. When used with the Invitrogen cloning kit, the M13 (-20) forward primer can be used as the sequencing primer.

The sequences obtained should be analyzed and compared to databases such as the Genbank or the EMBL, using the University of Wisconsin GCG software package. It is recommended to verify the differential expression by an alternative method, such as RNA blot hybridization, nuclear run-on or quantitative RT-PCR, due to the high number of false positive bands obtained in DD. However, we found that in many cases, less abundant transcripts seen after PCR amplification could not be detected by less sensitive techniques. For this reason we recommend to first clone and then sequence the PCR products of choice, so that verification can be done by standard quantitative PCR procedures. Most importantly, the DD method is not quantitative, thus only all or none band appearances should be further analyzed.

RESULTS:

In order to identify genes which are expressed specifically in the embryonic brain, total RNA was extracted from 50-100mg samples from whole brains obtained from mouse embryos (E14) or adults with the aid of RNAzolTM.. The RNA extracted from three individual animals was pooled and subjected to the differential display reaction above using the following primers:

It can be observed in Fig. 2 that at least 30 bands were clearly observed in each lane. It was found that some bands were embryo specific , e.g. expressed in the embryonic brain but not in the adult brain. Additional bands, found to be differentially expressed in part but not all duplicate reactions, may also be observed in the same figure. These were not considered for further study.

DISCUSSION:

Differential display of eukaryotic mRNAs has by now been employed as a research approach for almost three years in many laboratories5,7. It is a powerful technique which allows identification and cloning of those genes whose levels are subjected to changes upon specific circumstances. This technique enables us to detect genes whose RNA levels were changed. However, it is not possible to predict whether the levels of the corresponding proteins are actually modified. Since only a part of the transcript is amplified, the complete sequence data remains to be determined. Transcript amplification is not absolutely random, some RNA molecules will be preferably amplified over others according to their relative concentration and to whether they contain or not a sequence similar to the primer used for the DD.

Troubleshooting:

  1. Film blank or nearly blank: RNA may be degraded. Extract RNA avoiding degradation by RNAses (see section B.I), always check the quality of your RNA by gel electrophoresis and the concentration by spectophotometry. Verify that the labeled nucleotide used is the correct one (a and not g) and that it is not outdated. Some of the reaction components are especially unstable: e.g primers and nucleotides are very sensitive to pH fluctuations , check the acidity of the DDW used. The Reverse Transcriptase might be inactivated if kept for long periods on ice. Film exposure time may not be sufficiently long.
  2. Bands smeared Improper gel preparation and running; gels should be cast using fresh acrylamide solutions and should polymerize within 15 min of pouring, should be run at 40-55C.
  3. Number of bands too high or too low; Try a different primer, the pattern of obtained bands is variable (length and number) and depends on primer sequence.
  4. Results obtained are not reproducible; it is absolutely necessary to run all the DD reactions in duplicate and select for further characterization only those transcripts differentially displayed in both samples. Another fact to be taken into consideration is that strain or individual differences exist. Sample pooling helps to overcome this artifact. Differences can also be found between cells grown in different media or at different rates.
  5. No detectable product is obtained after band reamplification; use a sample from your PCR as template for a second round of PCR.

Alternative and Support Protocols:

The currently presented method is an optimization of the protocol developed by Welsh and colleagues6. It presents the following features as compared to the original protocol. Primer length affects both, the number of bands obtained in the DD reaction and the length of these products. We found that one 17-mer oligonucleotide combined with a higher dNTPs concentration, satisfied both requirements, the fragments obtained are relatively large (typically up to 600bp), and the number of bands is increased. Liang and Pardee3 have developed an alternative widely used method which was further optimized by their group and also by Bauer et. al.1 This protocol uses an oligo-dT anchored primer combined with a short (10-mer) arbitrary primer under low stringency conditions. However, using a single 17-mer primer allows transcript amplification at a random distance from the 3' end of the untranslated part of the mRNA (because the primer is not oligo-dT based), which increases the probability of obtaining meaningful sequence information from coding regions. Moreover, band length is typically higher. It permits, however the amplification of ribosomal and mitochondrial RNAs. The Welsh method is not sensitive to genomic DNA contamination. Because it includes two annealing steps at low temperature, so that priming in opposite directions will occur, genomic DNA will remain double stranded at this point, which will exclude it from further amplification.

In summary, this is a very sensitive and powerful approach for the detection and isolation of known and novel genes whose levels of expression are modulated in specific situations. Its importance is stressed for the analysis of nervous system gene expression, whose variability is enormous due to sequence complexity and the continuous dynamic changes enabling the fulfillment of the highly sophisticated functions, characteristic of nervous system cells.

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Mirta Grifman                   http://www.ls.huji.ac.il/~mirtag/home.html
Department of Biological Chemistry                      Tel: 972-2-6585450
Institute of Life Sciences                              Fax: 972-2-6520258
The Hebrew University of Jerusalem    e-mail:mirtag@leonardo.ls.huji.ac.il
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