amplify+specific+transcripts+from+RNA+3-18-13

-Plan --Develop conditions to amplify specific transcripts from RNA to determine if any of the final transcripts from my tumor library screening (see 8-26-12 Plasmids for verification) exhibit anything odd when I try to amplify the whole transcript from the 1st exon to the last exon. I will compare the results from tumor and normal RNA.

--Starting samples will be poly A purified samples.

--I can refer to the 2-1-13, 2-6-13 and 2-7-13 protocols to see how I first tried to amplify the transcripts. I can then try several different conditions to amplify a selected transcript. I will choose the Wfdc17 transcript to amplify so I will use RW17+FW17 primers to perform this optimization. I'll try a few starting RNA amounts, cycle numbers, and annealing temperatures. ---In the previous experiment I used 5 ng poly A RNA. I used 30 cycles with an annealing temperature of 55 C.

--In the new experiment I'll try 5, 50, and 100 ng poly A RNA. I'll try 25, 30, and 35 cycles. I'll try 50, 55, and 60 C annealing temperatures. see listing all combinations of conditions in an experiment

--Samples to use ---6-24-10 Tumor RNA ---Normal RNA from 1-29-13 (BALBsc mammary gland RNA)

--Primers to use ---RW17+FW17 and RW17Tr and FW17Tr (see Tumor Validation primers 1-28-13)

--Kits to use: PrimeScript RT and PrimeStar Max

-Expected size of final amplified transcript: 470 bp

-- 3-20-13 performed PCR with PrimeSTAR Max

Spreadsheet of samples found here: "C:\kurt\storage\CIM Research Folder\DR\2013\3-20-13\PCR\samples 3-20-13.xlsx"