IndirectDye-WordFile.docVIP

Microarray Indirect Labeling Probe Synthesis Protocol Written by Laura Hoopes of Pomona College August, 2003 The basis of this method is that the total RNA, containing polyA+ RNA molecules, is first annealed with OligodT (which is expected to anneal only with the mRNA having a poly A end) and cDNA is synthesized, with a reactive nucleotide (amino allyl dUTP) substituted for one of the regular nucleotides. No bulky dye is used in the cDNA synthesis, increasing yield. Afterwards, the cDNA is reacted with an activated form of the Cy dye and coupled to it, cleaned up from unincorporated dye, and hybridized to the array. One advantage to this method is that it is supposed to reduce dye asymmetry. The method is based on the method on the web site of Joseph DeRisi, as modified at Pomona College by Michelle Wu in Laura Hoopes’ laboratory, in consultation with Charles Kang at UCSF. In this protocol, use RNA paranoia: powder-free gloves, don’t get any dust into the samples, work on an RNase free surface, use RNase free tips and tubes, etc. Anneal Primers and Reverse Transcription:? Bring 50 ?g Total RNA to 14.5?l (by SpeedVac or adding more volume using DEPC H2O). Add 1?l oligo dT primer. ? Concentration ?l Oligo dT 5 ?g 1 Total RNA 50 ?g 14.5 ? Mix, quick spin Incubate at 70 °C for 10 min. Chill on Ice 10 min.? cDNA Synthesis To each reaction add 14.5 ?l of the following mix:? ? ?l 10.5 Reactions 10X buffer (stratagene) 3 31.5 50X aa-dUTP/dNTPs 0.6 6.3 DTT 0.1M 3 31.5 Stratascript RT 3 31.5 DEPC water 5 52.5 14.5 ?l Aliquot Mix, quick spin, then incubate at 42 °C for 2 hours.? This is a convenient place to stop in one laboratory period, since the unstable RNA has been copied into cDNA. The cDNA is not labeled with dyes yet so it is very stable and can be frozen for an indefinite period. The remainder of this protocol can also be done in one laboratory period of 3-4 hours. RNA Hydrolysis and Cleanup of the cDNA: 1 ?l 0.5 M EDTA pH 8 (add this