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. 2000 Jun 15;28(12):E62.
doi: 10.1093/nar/28.12.e62.

Improved NlaIII digestion of PAGE-purified 102 bp ditags by addition of a single purification step in both the SAGE and microSAGE protocols

J M Angelastro  1 , L P Klimaschewski, O V Vitolo
Affiliations

Improved NlaIII digestion of PAGE-purified 102 bp ditags by addition of a single purification step in both the SAGE and microSAGE protocols

J M Angelastro et al. Nucleic Acids Res. .

Abstract

Despite the success of microarray technologies, serial analysis of gene expression (SAGE) still remains the only technique that allows an accurate quantitative and qualitative analysis of cell transcription in a variety of physiological and pathological conditions. Nevertheless, the efficiency of SAGE is limited by the numerous gel purification steps required and these increase the possibility of contamination and reduce or inhibit the activity of the enzymes used in the protocol. In order to eliminate this problem, we have modified the original protocol by adding a single purification step before NLA:III digestion of the ditags. This allows us to increase the yield of digested ditags without reducing the amount of DNA or affecting the subsequent concatemerization.

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Figures

Figure 1
Figure 1
Polyacrylamide gels (12% PAGE in A–C, 8% PAGE in D), stained with SYBR Green I, that show the SAGE steps towards concatemerization. Using the original protocol leads to unsuccessful NlaIII ditag digestion in several cases, as depicted in (A) for naïve PC12 cells. Purification of ditags through a silica membrane rescues 102 bp ditags from the gel and increases the yield of 26 bp ditags, as shown in (B) for naïve PC12 cells and (C) for NGF-primed PC12 cells. This additional step combined with the heating step (6) allows formation of concatemers, as shown in (D) for NGF-treated PC12 cells. M1, 20 bp DNA ladder; M2, 100 bp DNA ladder.
Figure 2
Figure 2
SYBR Green I stained polyacrylamide gels (12% PAGE in A and B, 8% PAGE in C) showing the efficacy of the purification step in the microSAGE procedure for untreated PC12 cells. Prior to the use of Clontech spe10 spin gel filtration columns, 26 bp ditags are not detectable (A). As shown above in Figure 1 for SAGE, DNA purification through the columns remarkably improves NlaIII digestion (B) and facilitates formation of concatemers (C). M1, 20 bp DNA ladder; M2, 100 bp DNA ladder; M3, 1 kb DNA ladder.
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