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100 ng of samples with 260/280 readings of >1.8 and 260/230 readings of >1.9 were subsequently converted into cDNA using RT2 First strand kit according to the manufacturer's instruction.
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cDNA was generated from 500 ng of sample RNA using Omniscript RT (Qiagen, #205111).
Briefly 1000 ng of sample DNA was bisulfite treated using the Zymo DNA Methylation Kit (Zymo research, Orange, CA, USA).
For reliable performance of MLPA reactions, a minimum of 20 ng of sample DNA is recommended.
A total of 500 ng of sample DNA were used per assay.
A total of 100 ng of sample DNA was used per reaction.
All reactions were performed in triplicate with 5 ng of sample DNA for each reaction.
Successful fragmentation was achieved with 50, 125 and 250 ng of sample DNA (data not shown).
Subsequent assays using the TDP3 protocol and 200 ng of sample indicated 15/26 (58%) tumors were HPV positive.
All WT-Pico amplification reactions were performed with 2 ng of starting RNA whereas the IVT amplifications utilized 25 ng of sample RNA.
In brief, both 250 ng of reference aRNA and 500 ng of sample cDNA were collected in a single tube and kept dark.
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