matched DNA: RNA duplexes (on Northern Blots)and DNA: DNA duplexes (on Southern blots). This specificity is needed to be sure we are measuring the level of one particular transcript and that this is not contaminated with signal from closely related transcripts. RNA is isolated from cells, size fractionated on a gel; the thousands of mRNAs species form a smear on the gel which is punctuated by the strong ribosomal rna bands(28S and 18s that do not interfere with the analysIs. Image removed due to copyright reasons lease see http://www.accessexcellence.org/rcnl/gg/NuclEic.html --IGAPDH for one or Two gene prod abeled sequences specific Figure by MIT oCW Northern blots The breakthrough in developing microarrays for analyzing mRNa levels was to reverse the Immobilized mRNA population hybridized logic-instead of immobilizing the mRNAS for with labeled DNA probe representing one hybridization with one or two labeled or two genes complementary DNA (CDNA) probes, all possible CDNA probes are immobilized on a DNA Microarrays solid surface(usually glass slides ). The Immobilized DNA probes representing all spotting of probes is achieved robotically; the possible genes hybridized with labeled DNa probes are designed to specifically hybridize to only one nucleic acid sequence that represents a single mRNA species. The DNA Clones::::: thousands of DNa probes are dispensed from 96-well, or 384-well plates to an addressable site on the solid surface. the mrna population from each cell type purified and then copied such that the copy is fluorescently PCR amplification labeled. This fluorescent population is hybridized to the immobilized probes, and the robotic intensity of the fluorescence at each probe printing spot is proportional to the number of copies of that specific mRNA species in the original mRNA population hybridize target to microarray Figure by MIT OCWmatched DNA:RNA duplexes (on Northern Blots) and DNA:DNA duplexes (on Southern Blots). This specificity is needed to be sure we are measuring the level of one particular transcript and that this is not contaminated with signal from closely related transcripts. RNA is isolated from cells, size fractionated on a gel; the thousands of mRNAs species form a smear on the gel which is punctuated by the strong ribosomal RNA bands (28S and 18S) that do not interfere with the analysis. The breakthrough in developing microarrays for analyzing mRNA levels was to reverse the logic – instead of immobilizing the mRNAs for hybridization with one or two labeled complementary DNA (cDNA) probes, all possible cDNA probes are immobilized on a solid surface (usually glass slides). The spotting of probes is achieved robotically; the DNA probes are designed to specifically hybridize to only one nucleic acid sequence that represents a single mRNA species. The thousands of DNA probes are dispensed from 96-well, or 384-well plates to an addressable site on the solid surface. The mRNA population from each cell type purified and then copied such that the copy is fluorescently labeled. This fluorescent population is hybridized to the immobilized probes, and the intensity of the fluorescence at each probe spot is proportional to the number of copies of that specific mRNA species in the original mRNA population. Northern Blots Immobilized mRNA population hybridized with labeled DNA probe representing one or two genes DNA Microarrays Immobilized DNA probes representing all possible geneshybridized with labeled mRNA population Image removed due to copyright reasons. Please see http://www.accessexcellence.org/RC/VL/GG/nucleic.html Figure by MIT OCW. PCR amplification purification robotic printing hybridize target to microarray DNA Clones Figure by MIT OCW