HOME /

IVT Pipeline Overview

(Last updated: March 2020)

At the DRSC, we distribute reagents for RNAi screens from our library to users through three service pipelines: 

1)  Cherry pick: distribution of diluted PCR amplicons (DNA) 

PPMS → final DRSC IDs for worklist → cherry pick amplicons using MultiProbe → ship 

2)  Custom IVT (in vitro transcription): production of dsRNA based on a custom gene or DRSC ID list. The dsRNA is typically normalized prior to distribution. The dsRNA might be distributed in 96- or 384-well format, depending on user request.  

PPMS → final DRSC IDs for worklist → cherry pick amplicons using MultiProbe → PCR → QC via gel → gradient PCR on failed amplicons → QC via gel → IVT and clean-up → QC via Nanodrop and gel → normalization and re-array → stamp out assay-ready plates → ship

Notes: 

  • All molecular work will be in the 96-well format until we “stamp out” the assay-ready plates. This is when we use the Bravo to transfer samples from four 96-well quadrant plates into a 384-well assay-ready plate. Importantly, the layout of samples in the 96-well plates up to the normalization and re-array step is not the layout for the quadrant plates. 
  • The normalization and re-array step is where 1) the concentrations of each reagent are normalized across the 96-well plate, and 2) samples are moved into a particular layout in a 96-well quadrant plate for stamping out into assay-ready plates.

3)  Library or sub-library: distribution of the genome-wide library or sub-libraries (we currently have 14 sub-libraries). 

Workflow varies for each assay-ready plate in the library and is dependent on the current inventory in -80 freezers of specific assay-ready plates for each library. Some plates will require the entire IVT pipeline, while others may have normalized dsRNA plates that only need to be stamped out. There may even be assay-ready plates in the -80 freezers that only need to be shipped out—check the updated DRSC Freezer Inventory spreadsheet. 

Notes: 

  • The genome-wide library has assay-ready plates with all wells filled with samples, control reagents, or water.  
  • The sub-libraries have the outermost two rows and columns on the assay-ready plate filled with water instead of reagent. This is to mitigate any edge effects the assay-ready plate may experience. Thus, the number of samples we can fit on a 384-well plate is actually 240, including controls. 

Users generally conduct their assays in 384-well plates. Some users may want specific layouts, but we generally leave a two-well border of water in custom layouts as well. 

We have three main types of 384-well assay-ready plates. 

1)  “white solid” (Corning 3570): for luminescence assays. Inexpensive. This cost is included in our standard library distribution fees. 

2)  “black clear” (Corning 3712): for fluorescence or low-resolution imaging assays.  Inexpensive. This cost is included in our standard library distribution fees. 

3)  “Evotec/Opera” (PerkinElmer 6007558, “CellCarrier-384 TC”): for in-cell/other confocal or high-resolution imaging assays. The plates themselves are black and labeled “Greiner” (this is true for the current lot of plates, as of March 2020, and might not be true of future lots of these plates). Expensive and fancy; we pass the cost of these plates to users. 

Occasionally, users will have special assay-ready plates that they would like to use for their screens. They may provide their own, but oftentimes we order the special plates for them and charge them through PPMS. We have one of these special plates on hand as of March 2020: 

4)  “Evotec PDL-coated” (PerkinElmer 6057500): these are stored in the bottom shelf of the 4 degree fridge. 

We can normalize the concentrations of the reagents to whatever the users require, but in general we have two standard concentrations for dsRNA screens. 5uL of reagent is added to each well. 

For transfection: 16 ng/uL 

For bathing: 50 ng/uL 

We have a few common control reagents that are used for RNAi screening. Users can select the controls they want to use when placing an order through PPMS. Control target genes and the reasons for including these controls are summarized below. 

  • LacZ: negative control (there is no LacZ gene in flies). Should not affect cell health. 
  • thread (Diap1): Death-associated inhibitor of apoptosis. When this gene is knocked down, apoptosis is induced and cells start to die. A positive control. Cell death upon thread dsRNA treatment confirms that RNAi is working in general in the cells. 
  • GFP: Green Fluorescent Protein. For most assays, this is a negative control, as there is no GFP gene in flies. For assays based on GFP, this can serve as a positive control (GFP signal reduced upon knockdown with dsRNA targeting GFP). Our design does not target all GFP variants. In practice, we find that our GFP dsRNA impacts cell health more than our LacZ dsRNA does. 
  • Rho1: disrupts cell division, commonly leading to binucleate cells. Positive control (phenotype indicates that RNAi is working in general). This positive control is typically used for image-based assays.  
  • mCherry: knocks down mCherry expression (a red fluorescent protein) 

There are a few online management systems and databases that are used as part of the IVT pipeline. 

1)  DRSC-FGR instance of the Stratocore PPMS facility management platform (https://ppms.us/hms-drsc/login/?pf=2): accept and keep track of orders placed by users. Also used to manage user training, and for trained users to reserve time on instruments and cell culture hoods.

2)  FlyRNAi internal screening database and LIMS (https://www.flyrnai.org/cgi-bin/RNAi_login.pl): find location of amplicons, plates etc. This site manages our internal pipeline inventory and reagent information. It also manages data generated by screeners. Below is a summary of internal production processes tracked in FlyRNAi and are commonly used as part of the IVT pipeline.  

  • “Find wells for Amplicons and Genes”: This is the most commonly used function. It does exactly what it says on the tin—finding locations of reagents with DRSC numbers.
  • “Assay Plate Inventory”: enter the plate number to look up information about how many plates are available and when they were created. Useful when putting together a genome-wide library or sub-library order. 
  • “Assay Plate Check-Out”: be sure to check any assay-ready plates out of the system when shipping them to users in order to make sure the plate inventory is up to date. 
  • “Add New Aliquots to Database”: generate new plate names and labels when making fresh batches of assay-ready plates for the genome-wide library or sub-libraries. 
  • “Count Available Assay Plates”: gives an overview of the number of assay-ready plates that are available in the DRSC freezers. The database is not completely up-to-date however, so take any information here with a grain of salt. 
  • “Add New Assay Plate Types”: sometimes users want very specific assay-ready plate types that we do not normally carry. In this case, they may provide their own plates or ask us to order plates for them. Those new plates must be entered into the system for bookkeeping purposes. 
  • “Complete list of QC plates”: lists all the named 96-well plates in the database. Some quadrant plates are also included in this list; click on each plate name to see which reagents are located on that plate.

3)  UP-TORR (https://www.flyrnai.org/up-torr/): used internally to find DRSC IDs for reagents, used to select reagents for users who only provide a gene symbol. UP-TORR has acomplete record of DRSC dsRNA reagent templates at the DRSC.