RNAi Checklist

Submitted by on

The RNAi Experiment Annotation Checklist

This checklist contains a quick guide to the correct annotation of RNA interference experiments. For a more complete documentation of how to use the RNAi form see the RNAi experiment annotation guide(under construction). 

If you want a printed version  there is also a printer friendly version of the documentation.

Filling all the checkpoints will result in correctly annotated and reproducible experiments. The guide contains two sub checklists, one to complete at the time the experiment is started, and the second section to complete when the experiment is  completed and results are recorded. The RNAi annotation form is divided into several sub-forms by tabs as shown below. These tabs are treated in order of appearance, it is recommended to stick to the order of this checklist.

  1. Summary
  2. General
  3. Sample
  4. Target description
  5. Fragment description
  6. Validation
  7. Phenotype
  8. Upload

 

 

Checklist at Start of Experiment

 

Summary

  1. Description (Free text): mandatory field, a preliminary experiment description consiting of at least 10 words/ one sentence, in ENGLISH.

General

  1. Experiment Batch ID: Corresponds to Batch ID
  2. Experiment Contact: All Contact persons added
  3. Start date: Correct start date (of experiment, default is today, but that's not always correct)
  4. Experiment description: Concise intermediate experiment summary outlining the goal of the experiment, in ENGLISH.

Sample

  1. Organism: Correct organism? Most likely L. salmonis​
  2. Sex
  3. Number of individuals at start: Correct number of individuals counted.
  4. Developmental stage: field 1: at start of experiment
  5. Developmental stage: field 2: scheduled end stage at termination, if already known (recheck after termination for deviation)
  6. Sample protocol (optional): link sample protocol if available

Target description

  1. Primary Target ID: Mandatory field - correct target or multiple targets from the genome annotation selected. 
    1. Targets must be a either a transcript (EMLSAT) or gene EMLSAG, or a newly annotated nucleotide sequence. Do not use any other type of sequence feature (EMLSAP)!
    2. Number of annotated targets corresponds to fragment mixture (e.g. multi-target experiment contains a mixture of fragments)
  2. Gene or target symbol: Optional field for listing the required gene id, provide for easy search

Fragment description

  1. Fragment length: correct fragment length, identical to length of dsRNA fragment length 
  2. Fragment concentration: correct with respect to unit nano gram per micro-liter 
  3. One of Sense sequence or Antisense sequence: should be used only if using synthetic siRNA or other fragments or if the PCR product was sequenced. 
  4. Fragment ID: use if the fragment is stored in the genome annotation database as a Feature (like a gene or transcript, fragment sequence via New Feature).
    1. ​​ If Fragment ID used: check that Fragment sequence has valid Residues.
  5. Fragment Batch ID: corresponds to batch id assigned by fragment producer
  6. Fragment Label ID: must correspond to label written on the container that contains the dsRNA
    1. Correct format of Label ID: example F111, starts with F followed by up to 4 digits
  7. Primer names and Primer sequence: used for PCR-fragment produced dsRNA
    1. Correct number of primers: 2 or 4
    2. Correct primer sequence
    3. Primer sequences were checked for specificity (e.g. Blast, Primersearch)
    4. primer name: must indicate "forward" and "reverse" to determine primer direction
  8. Fragment producer: should be "Licelab" if produced in house
  9. Fragment producer contact: person responsible for fragment production can be contacted and has a valid LiceBase account

 

Checklist after Experiment Termination

  1. Re-visit all items on Checklist at Start for possible deviations, and incomplete annotation:
    1. Re-check General->Experiment desc​ription for preliminary description, in ENGLISH, checked for TYPOS
    2. Re-check Summary->Description (free text) changed description to final, in ENGLISH, checked for TYPOS. A good summary is comparable to a short abstract of a paper, contains a Results section.

 

Validation

  1. Efficacy of Knock-down: correct knock-down efficacy entered in percent compared to controll, if measured (by qPCR)
  2. Validation Protocol: optional, linked validation protocol 

Phenotype

  1. Number of Individual at End: correct number of counted surviving individuals or offspring at experiment termination
  2. Phenotype: correct and concise description of Phenotype, in ENGLISH, no TYPOS
  3. Quantitative phenotype: optional, check if applicable (e.g. growth curve), and provide measurements
  • -> Summary (Phenotype CV field is under Summary)
  1. Phenotype CV: correct annotation of phenotype, not <none>
    1. Missing phenotype annotated (No visible phenotype)
    2. Phenotype was annotated using existing terms as far as possible
    3. Multiple phenotypes have been annotated if multiple phenotypes have been observed
    4. Phenotype "Control" has been used to annotate control
      1. additional phenotype has been recorded correctly for Control  ("No visible phenotype"), and especially if unexpected (e.g. "survival->reduced") phenotype occurred

Upload

  1. Image File: all relevant raw images have been uploaded
  2. Gallery Image: A representative image has been selected for the gallery

Finally

  1. Preview: Preview has been used to check post: button 
  2. Save: Experiment has been properly saved: button  
  3. Experiment is listed in RNAi view
  4. Access control: checked proper accesss permissions under Access control (anonymous user checked = public!) button​ Access control