Diphenylamine–Aniline–Phosphoric Acid Reagent, a Versatile Spray Reagent for Revealing Glycoconjugates on Thin-Layer Chromatography Plates

✍ Scribed by Kimberly Anderson; Su-Chen Li; Yu-Teh Li

• Publisher: Elsevier Science
• Year: 2000
• Tongue: English
• Weight: 70 KB
• Volume: 287
• Category: Article
• ISSN: 0003-2697
• DOI: 10.1006/abio.2000.4829

✦ Synopsis

higher level in the benign pSV-R37 cell line than in all 3 of its malignant counterparts (Fig. 2B, middle). F3 was expressed at higher levels in the 3 malignant cell lines than in their benign counterpart (Fig. 2B,right). Analyses of the nucleotide sequences with an automatic sequencer (Model 373, Applied BioSystems, U.S.A.) confirmed that each band contained a single molecule. The sizes of these cDNA fragments are 382 (F1), 429 (F2), and 291 (F3) base pairs, respectively. Comparison of their sequences with those stored in the EMBL data library with BLAST revealed that the sequence of F1 did not correspond satisfactorily to any currently known gene. F2 represents a currently characterized gene, but its expression had not been previously associated with cancer metastasis. F3 is a segment of a currently known gene whose expression has been implicated in the malignant dissemination of cancer cells.

In comparison with the previously developed CCGE (7) and SDD (8) approaches, which require respectively 120 and 60 ng of total RNA, this new MDD approach requires only 2.5 ng of starting total RNA and perhaps much less. This method is therefore suitable for analyzing differential gene expression from RNA extracted from several hundred cells, making it an ideal general strategy for analyzing RNAs from scarce sources, such as those from small, microdissected lesions in pathological specimens. While CCGE is aimed specifically for the assessment of cDNA fragments within or near to the proteincoding region, both SDD and MDD can be used to perform similar analyses on the 3Ј terminal fragments when sufficient quantity of RNA is available. In the SDD approach, the 4-base-recognition enzyme MnlI (5Ј-CCTCN7/8-3Ј) is used to cut the doublestranded cDNA 8 , whereas in the MDD approach, the cDNA was digested with two 5-base-recognition enzymes. The MnlI generated cDNA fragments with the majority of sizes ranged from 100 to 450 bp, which is smaller than those generated by either one or two 5-base-recognition, type IIS enzyme(s) (Fig. 2A). Thus the cDNA fragments (ranged from 100 to 700 bp) generated by MDD can be distributed in a wider region of the denaturing gel and are better separated. Since the procedures used in SDD are simpler than those in MDD, we suggest that, while only MDD can be used to analyze the scarce RNA, the SDD method should be the first choice when sufficient quantity of RNA is available. However, digestion of the double-stranded cDNA with MnlI, which was used in the original SDD protocol, should be replaced by simultaneous digestion with two 5-base-recognition enzymes, as used here in the MDD protocol. This modification should further improve the SDD results.