![]() In the first step, mRNAs are reversely transcribed into cDNA, using a reverse transcriptase and an oligo-dT adapter as primer. The 3′ RACE is a well-described and optimized methodology, which exploits the natural poly(A) tail of mRNAs as a generic priming site for PCR amplification and takes place in two distinct steps. Since its establishment in 1988, RACE has emerged as the main strategy used to determine both the 5′ and/or the 3′ untranslated regions (UTRs) of any mRNA transcript, defining the transcription start point(s) as well as the poly(A) tail sites, accordingly. Rapid amplification of cDNA ends (RACE), also described as “one-sided” PCR or “anchored” PCR, is a molecular technique that enables the amplification of nucleic acid sequences from a messenger RNA (mRNA), between a specific internal region and either the 3′ or the 5′ end of the mRNA. This approach enables the broad and in-depth study of 5′ UTRs of any mRNA of interest, by offering a tremendous sequencing depth, while significantly reducing the cost-per reaction compared to commercially available kits. ![]() In this work we present an in-house developed 5′ RACE-seq method, based on the template-switching mechanism and targeted nanopore sequencing. Our results confirmed the existence of multiple annotated 5′ UTRs of the human KLK gene family members, but also identified novel, previously uncharacterized ones. As proof of concept, we implemented the described 5′ RACE-seq methodology to investigate the 5′ UTRs of several kallikrein-related peptidases ( KLKs) gene family members. Collectively, our results support the existence of two distinct 5′ termini for BCL2L12, being in complete accordance with the results derived from both direct RNA and PCR-cDNA sequencing approaches from Oxford Nanopore Technologies. We unveiled that the selection of hybrid DNA/RNA template-switching oligonucleotides as well as the complete separation of the cDNA extension incubation from the template-switching process, significantly increase the overall efficiency of the downstream 5′ RACE. The optimization of the described 5′ RACE-seq method was accomplished using the human BCL2L12 as control gene. In the current study, we developed a 5′ RACE-seq method by coupling a custom template-switching and 5′ RACE assay with targeted nanopore sequencing, to accurately unveil 5′ termini of mRNA targets. The implementation of the template-switching mechanism at the reverse transcription stage along with 5′ rapid amplification of cDNA ends (RACE) constitutes the most prominent and efficient strategy to specify the actual 5′ ends of cDNAs. However, 5′ ends of mRNAs are significantly underrepresented in these datasets, hindering the efficient analysis of the complex human transcriptome. The only difference between the V4 and V4+ is form factor, they both run the same OS and software.Technological advancements in the era of massive parallel sequencing have enabled the functional dissection of the human transcriptome. Even if you got a full V4+ kit with the fancy buttons and case, that's less than half the cost of a cirklon, and despite how fancy it looks it's not a very hard build - all the boards are simple and you could do the whole thing in one afternoon if you've got some DIY experience already. It would be really interesting to get to do a side by side comparison but that's never going to happen.Īlso I built my MBSeq in a fully wooden enclosure (other than front and rear panels) so my MIDI is warmer.ĮDIT: if you can DIY, a regular MBSeq V4 can be built for $250-$500 depending on the enclosure (if you have an aluminum panel milled that's about half the cost, I ended up going that way after a few unsatisfactory laser cut panels and it cost around $220 plus shipping for the thing, partly because it's milled on the front AND back and includes CNCed acrylic covers for the displays and everything the entire rest of the thing was somewhere in the $225-$275 range for PCBs and components - I did it a bit at a time so I don't know exactly but it wasn't much for what it is EDIT: I might be underestimating by about $75 now that I think about it, also I got the PCBs for the CV and trigger outputs and breakout boxes but haven't bought components or built them yet, nor the ethernet expansion so no OSC, network MIDI or WIFI connectivity). I've never used one but on paper the only thing that it has over the MIDIbox SEQ is the quality of the buttons and display in a standard MBSeq build.
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