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This is version 1.0_11711 of the Public TRC Portal, running on CodeIgniter version 1.7.2.

Frequently Asked Questions

Q: What is the difference between the intrinsic and adjusted scores for an shRNA?

The intrinsic score (also called "original score") of an shRNA assesses the target sequence for both predicted cloneability and knockdown performance, using an evolving set of rules. The scores reported in this website are calculated according to our most current ruleset ("Ruleset 9"), and have a maximum score of 15 for a hairpin with the best possible predicted cloneability and knockdown performance and a minimum of 0, for hairpins which exhibit sequence characteristics which could prohibit efficient cloning and/or degrade knockdown performance. We do not produce new clones with 0 intrinsic scores, but as our design rules have changed with time, there are older clones in the library which now have this score. The intrinsic score is not taxon- or target gene-specific, but is based solely on the sequence of the hairpin.

The adjusted score (also called "specificity score") is a function of the intrinsic score and two other factors which take into account the transcriptome of the target organism. The most important of these is the specificity factor, which penalizes hairpins with off-target matches (i.e. to genes other than the originally targeted gene). In absence of any off-target matches (specificity factor values greater than 1), the score is also increased slightly for hairpins which match the majority of the transcript isoforms of the target gene. The other contributor to the adjusted score is the miRSeed factor, which assesses the likelihood that the hairpin's microRNA seed region will exhibit unwanted microRNA effects, either due to sequence similarity with known miRNAs from the target organism, or to particularly common 7mer sequences in the 3' UTR regions of the organism's transcriptome. These factors are multiplied together with the intrinsic score to produce the final adjusted score, and again, higher scores are better. The specificity factor itself is generally not shown separately, except in the candidate hairpin design page for a given transcript, in which hairpin designs with specificity factor > 1 are given higher rank regardless of adjusted score.

Q: Why are there varying numbers of T's in the T runs at the end of the antisense strand of TRC haripins? Some have 4, some have 5 and some have 6 T's.

Over the course of TRC history, there have been a few changes in hairpin designs and the methods to produce clones. In terms of T runs, we started out designing 5 Ts as a Pol3 termination signal (4 Ts should do, but we added an extra). We produced two-thirds of the TRC1 library using oligos synthesized by IDT in a plate format. IDT oligos have good fidelity, and with this method we only accepted clones recovered with strictly 5 Ts.

Later we implemented a pooled cloning strategy, which uses an oligo pool (thousands of oligos in a pool) that was synthesized on a chip. This synthesis process has lower fidelity than IDT oligos, particularly in a run of mono-nucleotides. Our tests observed deletions in the run of Ts. So instead of 5 Ts, we put 6 Ts in each pooled oligo sequence. When hairpins were fully sequenced, we would still only accept clones which had perfect hairpin stem sequences, but as for the run of Ts, we accepted clones with either 5 or 6 Ts as good clones. So with this method, the run of Ts varies, with the majority at 6 Ts. While we do not yet explicitly annotate this difference among clones in the database, we will soon be doing so.