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Protocol of RNA Amplification

First strand synthesis

The standard protocol for amplifications employed a 10 µl reverse transcription (RT) reaction with 200 U SuperScript III (Invitrogen), I µl of linear acryl amide (0.1µg/µl, Ambion, ….) and 1 µg T7-N6s:  5’AATTGTAATACGACTCACTATAGGGNNNNN[N-Q]  in 1x first-strand buffer (Life Technologies) with a 50°C incubation.

  1. Add the following

RNA template (100 ng)

1 ug T7-N6s primer ,

  1. Incubate at 65°C for 5 min
  2. Transfer to ice quickly and incubate till reactions are cool
  1. Add the following to each tube

5 X buffer                                2 ml

10mM dNTP                           1 ul

0.1 M DTT                               1 ml

Rnase Inhibitor                        1 ml

1 ul of linear acryl amide (0.1ug/ul, Ambion)

add ddH2O to make a total of 10 ul for 100 ng,

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