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HARUO OHTANI
Negative
control
The negative control with cRNA of a sense orientation is important for the conformation of the specificity.
If the reaction products are the same between cRNAs of both
sense and antisense orientations, the following negative control
("competitive inhibition assay") can be used.
Transcribe non-labeled cRNA of antisense orientation as
shown below.
Add (with the total volume of 100/-ll)
Note: lO-fold larger amount of template DNA or RNA polymerase is not required for this reaction.
template DNA (l).lg/).ll)
2.5).l1
lOx transcription buffer
lO).l1
O.lM DTT
lO).l1
lOmM rATP
5).l1
lOmM reTP
5).l1
lOmM rGTP
5).l1
lOmM rUTP
5).l1
nuclease-free water
53).l1
RNase inhibitor
2.5).l1
RNA polymerase
2).l1
Tip the tube, incubate for 2hrs at 37°C.
Ethanol precipitation and sizing should be done in the same
way.
Do not add tRNA in the whole process. Measure RNA concentration at OD26o•
The negative control is done by adding lOO-fold larger
amount of non-labeled cRNA in the reaction solution containing
labeled cRNA with an antisense orientation.
If the reactivity is completely abolished, then further control
is to be done to confirm whether this abolishment of the reaction
is specific or not by adding the same amount of non-corresponding, non-labeled RNA. When this control does not affect the reactivity, then the results with the digoxigenin-Iabeled cRNA with