Author(s): Sanguinetti CJ, Dias Neto E, Simpson AJ
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Abstract A rapid silver-staining procedure for DNA fragments in polyacrylamide gels is described. The time required for band detection is 15 min and the limit of sensitivity 3 pg/mm2. PCR products subjected to this rapid staining protocol are readily recovered from the gel by excision and elution by incubation at 95 degrees C for 20 min. Bands of up to 3 kb have been recovered and reamplified from either recently prepared or dried gels. The rapid staining protocol significantly decreases the processing time required for silver-stained polyacrylamide gels, which is of particular importance in diagnostic situations. The recovery protocol allows individual bands from complex mixtures to be easily recovered for sequencing or probe preparation.
This article was published in Biotechniques
and referenced in Hereditary Genetics: Current Research
- Yosef Yarden
Classically, the 3âuntranslated region (3âUTR) is that region in eukaryotic protein-coding genes from the translation termination codon to the polyA signal. It is transcribed as an integral part of the mRNA encoded by the gene. However, there exists another kind of RNA, which consists of the 3âUTR alone, without all other elements in mRNA such as 5âUTR and coding region. The importance of independent 3âUTR RNA (referred as I3âUTR) was prompted by results of artificially introducing such RNA species into malignant mammalian cells. Since 1991, we found that the middle part of the 3âUTR of the human nuclear factor for interleukin-6 (NF-IL6) or C/EBP gene exerted tumor suppression effect in vivo. Our subsequent studies showed that transfection of C/EBP 3âUTR led to down-regulation of several genes favorable for malignancy and to up-regulation of some genes favorable for phenotypic reversion. Also, it was shown that the sequences near the termini of the C/EBP 3âUTR were important for its tumor suppression activity. Then, the C/EBP 3âUTR was found to directly inhibit the phosphorylation activity of protein kinase CPKC in SMMC-7721, a hepatocarcinoma cell line. Recently, an AU-rich region in the C/EBP 3âUTR was found also to be responsible for its tumor suppression. Recently we have also found evidence that the independent C/EBP 3âUTR RNA is actually exists in human tissues, such as fetal liver and heart, pregnant uterus, senescent fibroblasts etc. Through 1990âs to 2000âs, world scientists found several 3âUTR RNAs that functioned as artificial independent RNAs in cancer cells and resulted in tumor suppression. Interestingly, majority of genes for these RNAs have promoter-like structures in their 3âUTR regions, although the existence of their transcribed products as independent 3âUTR RNAs is still to be confirmed. Our studies indicate that the independent 3âUTR RNA is a novel non-coding RNA species whose function should be the regulation not of the expression of their original mRNA, but of some essential life activities of the cell as a whole.
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- Martha M Sklavos
Anti-MÃ¼llerian hormone deficiency in females with inherited bone marrow failure syndromes