RNA Ampification for Array Analysis阵列分析RNA扩增.docVIP

RNA Amplification for Array Analysis MessageAmp II? aRNA Kit Ambion Catalog # 1751 Date: SubID: Project ID: Contact Person: Genome ID: StepA: First-Strand cDNA Synthesis Start with 200ng-1ug total RNA or 20-100ng polyA RNA. Use 2ug total RNA or 1ug polyA RNA max. Most common to start with 1 ug of total RNA. Also, make sure you have 1 tube of universal reference per 4 sample tubes if the customer wants to hyb with our reference. Again, start with 1 ul reference or 1 ug/ul. Using table below, Mix RNA and T7 Oligo(dT) Primer, and bring the volume to 12 ?l. (pipette 1.1 uls of T7 Oligo dT primer and use digital pipette). Best to start with water, then add Oligo dT, then add sample Incubate 10 minutes at 70oC. (Use AMBION1 or AMP1 program in thermal cycler. Make sure to set tube vol to 100 uls because this is how much you will end up with at the end of the program. Also, choose Heated Lid.) Let samples go to 4oC for at least 2 mins before adding the Transcription Master Mix (2nd table down). Then take sample tubes out and spin 5 secs before adding mix… DO NOT VORTEX SAMPLE TUBES. Assemble the Reverse Transcription Master Mix at room temperature, then place on ice or in ice block while waiting for use. *Always keep the enzymes in the cold block (RNase Inhibitor and Reverse Transcriptase). Warm 10X FS buffer in hand or at RT and vortex or flick until precipitate is gone. When making this First Strand Mix allow for 1 more reaction than you need so you will allow for pipetting variation!! RT Master Mix 1 Rxn # rxns 10X FS Buffer 2 RNase Inhibitor* 1 dNTP Mix 4 ArrayScript* 1 Add 8 ul Reverse Transcription Master Mix to sample, mix by taping tubes on bench, flicking tubes 4-6 times and spin for 3 secs. Incubate for 2 hours at 42oC. Hold reactions at 4oC. StepB: Second-Strand cDNA Synthesis Assemble the Second-Strand cDNA Master Mix in order listed. *Keep enzymes in cold block at all times. Warm 10X SS buffer in hand or at RT an