FAQ: How much of the control RNAs should I use if I want to spike them into my sample?

If you intend to spike the control RNAs into your sample it is essential to use a much smaller quantity than when doing a control IP on them alone. This is because the m6A control RNA is highly modified and the antibody has a high affinity for it. This will result in the m6A control RNA becoming predominant in the sample after IP. Use of 1 µl of a 1:1000 dilution of each control RNA (0.1 fmol of each RNA) is recommended.  The control RNAs can be spiked in either before or after fragmentation of the RNA sample. If spiked in after, a concentrated stock of the control RNAs should be fragmented and then diluted prior to addition to the sample.