No abstract

Mammals compensate for different doses of X-chromosome-linked genes in male (XY) and female (XX) somatic cells by terminally inactivating all but one X chromosome in each cell. A transiently inactive X chromosome is also found in germ cells, specifically in premeiotic oogenic cells and in meiotic and postmeiotic spermatogenic cells. Here we show that the Xist gene, which is a expressed predominantly from the inactive X-chromosome in female somatic cells, is also expressed in germ cells of both sexes, but only at those stages when an inactive X chromosome is present. This suggests support for the putative role of Xist as a regulator of X-chromosome inactivation and suggest a common mechanism for the initiation and/or maintenance of X-chromosome inactivation in all cell types.

No abstract

The reverse transcriptase-polymerase chain reaction (RT-PCR) provides an effective m e t h o d for detecting very small a m o u n t s of a specific mRNA in a small sample of total RNA.O,2) Unfortunately, for purposes of detecting RNA by this procedure, after the initial step of converting RNA i n t o cDNA using reverse transcriptase, the multiple rounds of amplification catalyzed by DNA polymerase are equally effective at a m p l i f y i n g either the cDNA or cont a m i n a t i n g g e n o m i c DNA. Even minuscule a m o u n t s of c o n t a m i n a t i n g DNA (<1%) can produce a falsepositive amplification signal in an RT-FIGURE 1 Effects of pretreatment with nuclease on production of amplification signals by RT-PCR. Five hundred nanograms of total RNA from mouse liver supplemented with 0, 1, 5, or 10% genomic DNA was subjected to RT-PCR using primers determined from the mouse 13-actin cDNA sequence, (14) and reverse transcriptase and AmpliTaq DNA polymerase (PerkinElmer Cetus). Primers used were: (upstream) 5'-GCGGACTGTTACTGAGCTGCGT-3' and (downstream) 5 ' -GAAGCAATGCTGTCACCTTCCC-3 ', which delineated a 453-bp amplification product from either a cDNA or genomic DNA template, since this sequence apparently does not span an intron in genomic DNA. In each case a comparison of amplification with (+) or without (-) the addition of reverse transcriptase is shown. PCR Methods and Applications 279Cold Spring Harbor Laboratory Press on June 7, 2019 -Published by genome.cshlp.org Downloaded from Technical PCR reaction (Fig. 1). One method to distinguish amplification signals templated by genomic DNA versus mRNAderived cDNA is to use primers that span an exon-exon junction in the cDNA, such that the presence of an intron in the genomic DNA template will yield a larger amplification product than that emanating from the intronless cDNA template. (3) However the necessary sequence information to design such primers (i.e., location of introns) is not always known for a particular gene, and some functional genes lack introns, as do all processed pseudogenes. Thus, it is often desirable to be able to eliminate contaminating genomic DNA directly from samples of RNA prior to RT-PCR.DNA can be eliminated from RNA samples by treatment with RNase-free DNase I as a separate step in the purification of total RNA. (4-6) However RT-PCR is often used to analyze very small samples of total RNA, and attempts to recover such small amounts of RNA following a separate DNase digestion step can result in the loss of all nucleic acid, including RNA. Recovery can be augmented by addition of carrier RNA, but this introduces another potential source of amplification artifact. We have developed a one-step procedure for the removal of contaminating genomic DNA from RNA samples that can be carried out in the same tube as the subsequent RT-PCR reaction, without addition of carrier RNA. We show here that this procedure can effectively remove even very large amounts of contaminating genomic DNA, with no appreciable loss of RNA. METHODSRT-PCR was perf...