Cherry pick amplicons

(Last updated: March 2020)

We offer to users the option to request PCR templates from our collection. Users will receive these, amplify them, and use them in their own IVTs to generate dsRNAs. This is typically done when a user is interested to test just a small number of reagents. But we have also received larger orders (when larger orders come in, we would typically make sure they know about the custom IVT option, and some users decide to do that instead of just receiving a cherry pick). 

Users often assemble their own list of reagents using our database when they place an order. In that case, the DRSC number is provided. However, sometimes users select “Please assist with amplicon selection” on the PPMS order service form. In this case, they will generally provide the gene name(s) and it’s up to us to select appropriate designs for them. We use the UP-TORR tool (https://www.flyrnai.org/up-torr/) to find up-to-date info about which reagents are available from our library. Note that users sometimes list controls in their list but also have the option on our PPMS request form to click to add specific control templates. If a user is placing a request for the first time and does not indicate any controls, we should contact them to make sure they understand the usefulness and availability of controls. Sometimes, after such an email conversation with a new user, controls are added to the request. 

Selecting amplicons using UP-TORR (when the user provided a gene list) 

1)  On the PPMS order service form, scroll past the list of genes to “For each gene, would you like to receive” and see what the user selected.  

  • Users can choose to pick all the available unique designs for a gene, two or three unique designs (if available), or exactly two unique designs. We generally have two to three unique designs per gene in our library. 

2)  Once you know how many designs per gene the user wants, copy the list of gene names. Go to the UP-TORR site and paste it into the text box under “Enter Information.” 

3)  On the left-hand side of the page under “Input Options,” make sure “Gene,” “Gene Identifier,” and “DRSC dsRNAs” are selected. Uncheck all other boxes in the “Gene” section. Leave everything else on the page in their default settings. 

4)  Hit the “Submit” button on the right-hand side of the page. 

5)  A large table will pop up in a new tab. Hit the “Download Excel” button near the top of the page and open the downloaded spreadsheet. Make sure to save it to your computer. 

6)  There are a few criteria to pay attention to when selecting designs.  

  • OTE (off-target effect): all designs with a value >5 are excluded. Place a filter on the column by highlighting the entire column. On Excel’s Home tab, find “Conditional Formatting”  “Highlight Cell Rules”  “Greater Than…” Enter “5” and click “OK.” All designs with OTE >5 will be highlighted red. 
  • Target: the ideal designs have “Target 1 gene, all isoform(s).” Double check if this is indeed the case using the “Isoforms” column. If the design doesn’t target all isoforms, choose another design if it’s available. 
  • Region: CDS > 5’UTR > 3’UTR. If the design overlaps the CDS and an UTR, check to see which region is most of the design located. 
  • Seq Len: longer designs are generally preferable to shorter designs. However, if a shorter design is better in other ways that a longer one (ex. longer design is in the UTR while shorter design is in the CDS), use the shorter design. 

Choosing designs is ultimately a judgment call. We want to provide the number of designs that the user requests, so sometimes a not-ideal design will still be included if our library only has a couple designs for a specific gene. 

7) Once the list of designs (DRSC ID list) is decided, check in with the user to obtain their approval for the ID list. Mention if there are fewer designs for a particular gene that they requested and remind the user that they can use UP-TORR to see the available designs for each of their genes of interest. Make changes as necessary to the ID list if the user has specific requests. When in doubt, check in with the DRSC director. 

8) Once the user approves the ID list, move onto the PPMS orders & cherry picks protocol for instructions on how to find the locations of the reagents. 

  • If there are no dilute PCR amplicons for a particular design (ex. the well is empty, the plate is not available), go back to the original list of all designs and see if there are other designs available for that particular gene. 

9) Users will generally request that controls be included in their orders. There are dilute PCR amplicons for all four controls (LacZ, thread, Rho1, GFP) in Eppendorf tubes in the PCR box (“Kasia PCR” in the -20 Freezer #2). However, the controls are also included on actual plates in the freezers as part of the reagent library, so they can also be retrieved via cherry pick. Just look up the DRSC IDs for each control. There should be multiple designs for each control as well.