Table 1 easyCLIP troubleshooting.
From: easyCLIP analysis of RNA-protein interactions incorporating absolute quantification
Step | Verification |
|---|---|
RNAse digestion | Verified by fluorescence on nitrocellulose (NC): under-digested sample runs at the top of the gel and over-digested sample has no smear upward in the minimal region. |
5′ and 3′ ligations | Assessed by protein shifts and total fluorescence on NC. With more difficulty, the RNA and adapter shift methods are usable for exact determinations if the protein shift pattern is too complex. For RNA and adapter shifts, it is best to have library sizes >10 fmol. |
Nitrocellulose to reverse transcription (RT) | The extraction from NC and oligonucleotide(dT) purification can be assayed by dot blotting the oligonucleotide(dT) eluate on nylon. Purified RNA must contain the L3 adapter, so fluorescence from the L5 adapter corresponds to the completed library. |
Reverse transcription | Verified by comparison of qPCR amplification with input to RT. PCR reactions amplify more quickly the more complex the input sample. The L5 adapter fluorescence input to RT corresponds to the completed library. If L5 fluorescence input is ~1000 s, the input is in the femtomolar range, which should be visibly in the exponential PCR phase before cycle 14. It should never be necessary to amplify >~16 cycles. Completed libraries before RT can also be visualized by the RNA shift method. |
Amount of input RNA | Determination of the 5′ ligation efficiency by any method and a quantified standard to translate fluorescence values into RNA molecule numbers allow for a determination of the total amount of input RNA. |
