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===Prep=== * RT primers must be diluted to appropriate concentrations depending on the input: ** For 1 ng of total RNA, use 0.5 ul of 32 pmol/ul. ** For 100 pg of total RNA, use 0.5 ul of 3.2 pmol/ul. ** For 10 pg of total RNA, use 0.5 ul of 0.32 pmol/ul. * See diluted RT primer layout under Arichard:Reagents. ====Dilute total RNA to 2 ng/ul, on ice==== # Add 1 ul 100 ng/ul RNA to 9 ul H2O --> 10 ul 10 ng/ul RNA. # Add 2 ul 10 ng/ul RNA to 8 ul of H2O --> 10 ul 2 ng/ul RNA. * Store all at -80 degC. ====Dilute ERCC RNA Spike-in Control to 1:10,000, on ice==== # Add 1 ul of undiluted ERCC to 9 ul H2O --> 10 ul 1:10 ERCC. # Add 1 ul of 1:10 ERCC to 9 ul H2O --> 10 ul 1:100 ERCC. # Add 1 ul of 1:100 ERCC to 9 ul H2O --> 10 ul 1:1000 ERCC. # Add 1 ul of 1:1000 ERCC to 9 ul H2O --> 10 ul 1:10000 ERCC. * Store all at -80 degC. ====Mix total RNA and ERCC RNA Spike-in Control==== # Add 5 ul 2 ng/ul RNA to 2 ul 1:10000 ERCC --> 7 ul 1.42 ng/ul RNA 1:35000 ERCC. ## Add 0.7 ul of this to 0.5 ul 32 pmol/ul primer for 1 ng total RNA samples. ## Need total of 2 x 0.7 ul + 2 ul for dilution to 142 pg/ul RNA 1:350000 ERCC = 3.4 ul. # Add 2 ul 1.42 ng/ul RNA 1:35000 ERCC to 18 ul H2O --> 20 ul 142 pg/ul RNA 1:350000 ERCC. ## Add 0.7 ul of this to 0.5 ul 3.2 pmol/ul primer for 100 pg total RNA samples. ## Need 14 x 0.7 ul + 1 ul for dilution to 14.2 pg/ul RNA 1:3500000 ERCC = 10.8 ul. # Add 1 ul 142 pg/ul RNA 1:350000 ERCC to 9 ul H2O --> 10 ul 14.2 pg/ul RNA 1:3500000 ERCC. ## Add 0.7 ul of this to 0.5 ul 0.32 pmol/ul primer for 10 pg samples. ## Need 12 x 0.7 ul = 8.4 ul.
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