PCR Products

When analysing PCR products, I see a band corresponding to the size of primer dimers, especially in the sample that was cut low from cDNA gel.

Yes, it is common to see this band in the sample that was cut low from cDNA gel, and sometimes also in other samples. This is due to contamination from short cDNAs that only contain the sequence of RT primer. If this primer dimer is the dominant product on gel, we advise against sequencing the corresponding sample.

At the end of the procedure, I didn't understand if you purify the final PCR product or if the PCR product is ready for sequencing?

PCR products are ready. The gel is just for quality control. Alternative methods (bioanalyser) are also possible.

In the protocol, is the final PCR product quantified before submitting?

Yes, the PCR product needs to be quantified. We use both qPCR and bioanalyser.

Can I gel purify PCR products and re-PCR using the same primers?

Normally, the products of the first PCR should look clean on the gel, otherwise it is a sign of a library that is of low complexity, and is unlikely to generate informative data. Therefore we advise against re-PCR, but it can be done as the last resort.

After final amplification of the library and gel extraction, there are several distinct bands, and A260/280 indicated there were good amount of DNAs. However, QC results (capillary electrophoresis) provided by the sequencing company indicated there were no peaks in some (if not all) samples (a few samples gave a very sharp peak at ~120bp, but they say “NA” in the section of sample concentration). Since they charge by lanes, not by number of samples, for NGS analysis, I wonder if I should include these samples for sequencing while trying to obtain better samples (quality/quantity) in the second try.

I advise against proceeding with the poor samples. 120bp peak is primer dimer. If you overamplify your library, there may be additional peaks on the gel at higher MW, but these would be just additional artefacts. If you mix with good samples, they will just produce useless reads that won’t map. Make sure you follow the quality control steps described in Huppertz et al, Methods 2014 - especially comparing the PCR products obtained after cutting from different sizes of cDNA gel - the sizes of these PCR products should agree with where cutting was done.

We wonder if the iCLIP protocol could give problems with small RNAs, as truncated fragments may be too short to harbour useful information.

We find that if a protein crosslinks to small RNAs, these will be the dominant sequence species in the resulting data. In such case, there will be enough reads for these.

I have been trying a variation of iCLIP, the irCLIP protocol by Zarnegar et al., and came across your google document with questions and answers. First of all, it is very useful, thank you very much. Secondly, I have tried the protocol with one of the hnRNP proteins and after the library preparation (which I think follows Flynn et al., Fast-iCLIP), I see a strong peak at 148 bp and a fainter smear ranging from ~120-200 bp. The empty PCR products are reported to be 137 bp. I was wondering if this pattern is normal to be seen by bioanalyzer and if not, have you ever experienced a discreet stronger band in your iCLIP final libraries.

You are right, due to the longer barcodes, the primer dimer band migrates around 137 bp in the irCLIP protocol. I’m unsure what the 148 bp band would be, but it may also be a type of an artefact - usually strong peaks are artefacts. We tend to have a problem with primer artefacts when using the irCLIP protocol, so gel purification is a must. In your case, I guess purification of signal >155bp would probably be best.

I have been using the iCLIP protocol modified with the irCLIP protocol. After the amplification with the solexa primers I have run my cDNA library on a TBE gel and cut between 155bp and 400bp. When I took the samples for analysis of the tape station what I am seeing is in the picture below. I am not sure if the samples are good enough to send for sequencing or if I have some primer dimers in this. Could I please have some advice on how to proceed please?

The sizes of the first library look a bit unusual (very long products with peak at 272, but are you sure this size is right? - it seems to me that this ‘272’ peak is same as the 139 peak from the 2nd library), but probably fine, but in the 2nd library the peak at 139 are indeed primer artefacts (these we find particularly problematic with irCLIP). So a 2nd gel purification for the 2nd library would be good where you again cut above 155nt (it seems that with the 1st purification you didn’t manage to get rid of the artefact well). If the two libraries have different barcodes, you can possibly mix them before you load the gel again to repurify.