CastAway precast gels for fast analysis of RAP-PCR and differential display
Douglas McKenzie and Douglas Drake
Stratagene Cloning Systems, Inc.
Stratagenes new CastAway precast sequencing gels were used to detect and isolate differentially expressed gene products amplified by the RNA arbitrarily primed polymerase chain reaction (RAP-PCR). A human myelomonocytic cell line (HL60) was stimulated for 4 hours with phorbol myristate acetate (PMA). Arbitrary primers were used for first-stand synthesis and PCR amplification of the cellular mRNA. The PCR products were run on the CastAway system, and the pattern of PCR bands was compared to the pattern from a nonstimulated control. Fourteen unique bands representing differentially expressed gene products due to the PMA stimulation were identified, isolated from the gel and reamplified by PCR for further analysis. Differential expression was confirmed by Northern blot analysis or reverse Northern blotting.
Differential gene expression provides the basis for many fundamental cellular processes associated with differentiation and development. The identification and cloning of genes whose expression is modulated is an important step in advancing our understanding of these phenomena. Recently, two different PCR-based techniques were described that provide both the means and the power for analyzing gene transcription on an extensive scale. These techniques, referred to as RNA arbitrarily primed polymerase chain reaction (RAP-PCR)1 and differential display (DD),2 share several features. Both techniques rely on the degenerate binding of arbitrarily chosen primer(s) to sample multiple cDNA species during the polymerase chain reaction (PCR) and, following adequate amplification, yield a unique fingerprint reflective of these species. Although the two techniques were originally quite distinct, re cent changes in the conditions used during DD have made DD closer in nature to RAP-PCR.3 The PCR products generated in RAP-PCR and DD are subjected to gel analysis using high-resolution polyacrylamide gel electrophoresis (PAGE). Bands of interest are excised from the gel, reamplified, cloned and sequenced. Differential expression of the cloned gene product is verified using Northern or reverse Northern blotting.4
Because of the versatility of these approaches for identifying differentially expressed genes, the most difficult and time-consuming portion of the techniques is the ultimate characterization of the gene product. In this article, we examine the utility of Stratagenes new CastAway prepoured sequencing gels for analyzing PCR products. Using CastAway gels eliminates tedious pouring of polyacrylamide sequencing gels and makes analysis of RAP-PCR or DD faster and easier. The thin, 0.25-mm CastAway gels can be used to quickly run samples, identify differential gene products of interest and isolate and purify band fragments from the gel.
The CastAway precast system and sequencing gels were used for analysis of several RAP-PCR products. The system under investigation consisted of a human myelomonocytic cell line (HL60) that was stimulated for 4 hours with the tumor promoter agent, phorbol myristate acetate (PMA) at a final concentration of 16 ng/ml. We sought to detect immediate-early genes whose expression was modulated in response to this treatment. Messenger RNA was isolated from the control (nonstimulated) cells and the PMA-stimulated cells and was used in RAP-PCR according to the protocol provided with Stratagenes RAP-PCR Kit. Three different arbitrary primers were used for three separate first-stand cDNA synthesis and further PCR amplification reactions in the presence of [a-32P]dATP. Following PCR amplification, an aliquot of the RAP-PCR products obtained with each cell population was resolved for 2 hours at 50 watts on a CastAway prepoured 6% polyacrylamide gel. The gel was fixed and dried according to the protocol provided with the product. The dried gel was exposed to film overnight and developed. The RAP-PCR fingerprints obtained with each of the arbitrary primers are shown figure 1.
We identified 14 different PCR products whose expression was modified as a result of PMA stimulation. The expression of 3 of these products was down regulated in response to PMA, whereas the expression of 11 products was elevated. Primers 1 and 2 yielded differential gene candidates, while none were observed with primer 3. The size of the products ranged from 100 to 550 bp. The gel was reexposed to a second film, and candidate bands were outlined with ink and given an identifying number on the second film figure 2. The dried gel was rehydrated in water overnight and then placed on the second film. Glogos II autorad markers that had been affixed to the glass plate prior to the second exposure provided the means for aligning the bands marked on the film with the corresponding region of the rehydrated gel. A pipettor bearing a 1000-l aerosol- resistant tip was used to repeatedly aspirate the gel sections at the positions corresponding to the band of interest. The pipet tip containing the aspirated gel section was placed into a microcentrifuge tube along with 50 l of 5 mM Tris-HCl, pH 8.0 and 0.1 mM EDTA and was incubated at 65C for 2 hours. The pipet tip was removed, and the tube was centrifuged for 5 to 10 seconds in a microcentrifuge. Supernatants from the eluted gel slices were used as templates in PCR amplifications using the original RAP-PCR primers. The reamplified RAP-PCR products were analyzed by agarose gel electrophoresis figure 3. Products of appropriate size were obtained with all 14 samples. To verify the efficacy of the fragment isolation by aspiration, the gel plate was dried and exposed to film for a third time (data not shown). Silver staining was missing from the film at positions corresponding to the eluted bands, confirming that aspiration had effectively removed the band of interest from the hydrated gel.
Techniques such as RAP-PCR and DD have wide application because of their ability to detect global changes in gene expression. However, both approaches require substantial time and effort for analyzing the identified gene products. Stratagenes new CastAway precast sequencing system can be used to expedite the process by reducing the time and effort necessary to detect and isolate differentially expressed gene products.
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