Background Serial analysis of gene expression using small amounts of starting
Background Serial analysis of gene expression using small amounts of starting material (microSAGE) has not yet been conclusively shown to be representative, reproducible or accurate. of linkers used to generate ditags. While it offers been shown that standard SAGE is definitely representative and accurate , the same has not been clearly demonstrated for microSAGE. Moreover, the degree to which data generated by either SAGE or microSAGE are reproducible, in the sense of giving identical tag representation from duplicate samples, has not been thoroughly CDKN2AIP resolved. Given the time and expense involved in building and sequencing SAGE libraries, concerns about how reproducible SAGE data are in general, as well as how representive and accurate microSAGE data are in particular, may deter potential users from going after this approach. We have resolved these questions by building and analyzing a series of microSAGE libraries. We made libraries from your same mRNA samples to examine the variability due to library construction, and made sublibraries from identical swimming pools of ditags to examine the variability due to the last methods in SAGE library construction, as well as due to sampling. We have also made and analyzed libraries from cells from both age-matched and non-age-matched individuals to examine the variability due to cells preparation as well as individual variations in gene manifestation. We found that microSAGE data, as previously demonstrated for standard SAGE data , are highly accurate in the sense that they reflect known cells and developmental gene-expression profiles. We found that 20675-51-8 manufacture tag distribution is virtually identical in samples constructed from either identical mRNA samples or ditag ligation reactions. However, we found large variations in tag distributions between both age-matched and non-age-matched human being peripheral retinal libraries. We even saw a relatively large variation in tag distributions between libraries made from cells pooled from small numbers of individual neonatal mice, a fact that may reflect developmental asynchrony as well as baseline individual variance in gene manifestation. This truth offers implications for any expression-profiling approach, and suggests that in order to average out individual variations in gene manifestation, many different samples of 20675-51-8 manufacture a cells of interest will have to be examined. Results MicroSAGE data are representative and accurate To determine whether microSAGE data are representative, we carried out a virtual Rot analysis. A virtual Rot analysis, much just like a Rot analysis generated by measurement of mRNA reassociation kinetics, examines the relative portion of total mRNA made up of transcripts of high, medium or low large quantity. It is constructed from SAGE data by cumulatively plotting the total fraction of all tags displayed by tags of a given large quantity level. Virtual Rot analyses of adult mouse whole retina and adult hypothalamus showed a distribution that closely reflects an actual Rot analysis of mouse mind RNA (Number 1a,1b,1c) . Number 1 Virtual Rot analyses. (a) Virtual Rot analysis of hypothalamus library A. This plots the cumulative portion of tags in the library against the total tags present for each tag-abundance level. The boundary between moderate- and low-abundance transcripts … To determine whether microSAGE data can provide an accurate measure of mRNA large quantity – that is, give tag levels that match levels of mRNAs for which abundance levels possess previously been measured – we examined our adult mouse retinal library 20675-51-8 manufacture for tags related to several such mRNAs. Although no complete 20675-51-8 manufacture quantification of mRNAs in mouse retina has been made, 20675-51-8 manufacture measurement of rhodopsin, beta-tubulin and interphotoreceptor retinol-binding protein (IRBP) mRNAs in adult rat retina indicate that these represent 1.25-2.5%, 0.2% and 0.1-0.2% of total RNA, respectively . These figures are based on the observation that mRNA consists of roughly 2% of total RNA in both rat and mouse retina (S.B. and C.L.C., unpublished work). In the mouse, we found that rhodopsin displayed 2.1%, beta-tubulin 0.08%, and IRBP 0.13% of total SAGE tags, values that on.