Opened 6 years ago

Closed 6 years ago

#672 closed task (fixed)

RNA concentration normalization wizard

Reported by: Nicklas Nordborg Owned by: Nicklas Nordborg
Priority: critical Milestone: Reggie v2.18
Component: net.sf.basedb.reggie Keywords:
Cc:

Description (last modified by Nicklas Nordborg)

A new step that is performed immediately after RNA extraction and Nanodrop concentration measurements.

Instead of putting the extracts RNA in the freezer as is, an aliquot is taken and diluted to the concentration that we need in the current mRNA step. The remaining RNA is put in the freezer while the aliquot is stored temporarily since it should go into library preparation very soon.

In theory, if only such pre-diluted aliquots were used in the mRNA wizard the lab protocol would have only zeroes in the H20 column. The RNA dilution registration would not have to be used.

Also note that the mRNA wizard require that RNAQC items exists and have either RQS or RIN values. In a "real-time" scenario this will not be the case, so the wizard must work without those values.

Summary of changes

  • At the end of the 'Create aliquots on Bioanalyzer/Caliper? plates' wizard it should be possible to enter the 'RNA concentration normalization wizard'. The current RNA items are automatically selected (the wizard should support manual selection as well).
  • RNA items with too low quantity are not normalized, but flagged with NotEnoughRemainingQuanity immediately.
  • It should be possible to print a lab protocol with dilution volumes and place for comments.
  • Registration is done as the last step and should create new items with '.a' as last part of the name and subtype=RNANormalizedAliquot. The RNA items are annotated with AutoProcessing=Disable so that the create mrna wizard doesn't select them.
  • Labels for the tubes should be printed from the partion wizard together with the other labels.
  • The mrna wizard should support selecting normalized aliquots. Mixing normalized with non-normalized RNA is not supported. Biggest difference is the underlying query used for automatic selection and that it should be possible to select items without any RQS or RIN value.
  • The 'Lab protocols for mRNA and cDNA preparation' need to support the normalized items. No dilution is needed and storage box is temporary.
  • 'RNA dilution registration' wizard doesn't have to be done on the plate.
  • The rest of the downstreams wizards should be the same, but they probably have to be made aware of the extra item that may site between mRNA and RNA.

Change History (17)

comment:1 Changed 6 years ago by Nicklas Nordborg

Description: modified (diff)
Summary: RNA dilution wizardRNA concentration normalization wizard

comment:2 Changed 6 years ago by Nicklas Nordborg

Status: newassigned

comment:3 Changed 6 years ago by Nicklas Nordborg

(In [2898]) References #672: RNA concentration normalization wizard

Created new subtype RNANormalizedAliquot and selected annotations for it (DilutionDate, DilutionOperator and NdConc).

comment:4 Changed 6 years ago by Nicklas Nordborg

(In [2899]) References #672: RNA concentration normalization wizard

Add *.r.a label to the label file.

comment:5 Changed 6 years ago by Nicklas Nordborg

(In [2900]) References #672: RNA concentration normalization wizard

Updated 'DNA/RNA/FlowThrough registration' to include a checkbox for 'Pre-normalize' RNA. The checkbox is active and automatically selected for all RNA items with enough quantity (>1.1 µg). The annotation is temporarily saved on the RNA item to indicate that it should be selected for normalization.

comment:6 Changed 6 years ago by Nicklas Nordborg

(In [2901]) References #672: RNA concentration normalization wizard

Updated 'DNA/RNA/FlowThrough registration' so that RNA with too little remaining quantity are flagged (NotEnoughRemainingQuantity) and added to the Flagged RNA list.

comment:7 Changed 6 years ago by Nicklas Nordborg

(In [2902]) References #672: RNA concentration normalization wizard

Remove PreNormalize annotation again and replaced it with a new value (PreNormalizeRNA) for the AutoProcessing annotation.

comment:8 Changed 6 years ago by Nicklas Nordborg

(In [2903]) References #672: RNA concentration normalization wizard

Added "RNA concentration normalization" wizard.

By default it will select RNA annotated with AutoProcessing=PreNormalizeRNA. It is possible to manually select other RNA.

The second step will display a dilution protocol for the selected RNA items. It checks that there is enough remaining quanitity (>1.1µg). In this step it is possible to print a lab protocol.

The last step is for confirming that the normalization has been done and for entering a date and operator annotation.

When registering new RNANormalizedAliquot items will be created. Quanitities and concentration values are automatically calculated (we re-use the NDConc annotation to store the new concentration).

comment:9 Changed 6 years ago by Nicklas Nordborg

(In [2904]) References #672: RNA concentration normalization wizard

Added a count on the index page for counting pre-normalized RNA waiting for mRNA.

Set creation date on normalized RNA.

comment:10 Changed 6 years ago by Nicklas Nordborg

(In [2905]) References #672: RNA concentration normalization wizard

Aliquots should use .n suffix instead of .a

comment:11 Changed 6 years ago by Nicklas Nordborg

(In [2906]) References #672: RNA concentration normalization wizard

Started with the "Create new mRNA plate" wizard and the rest in "RNA to cDNA wizards" section.

  • The "Auto-select" dialog has an option if regular or pre-normalized RNA should be selected. In the latter case, no filters can be used except start date.

  • Manual selection include pre-normalized RNA in the filter.

  • Missing location and RQS/RIN are ignored for pre-normalized RNA.

  • It should be possible to mix regular RNA with pre-normalized RNA. If only pre-normalized RNA is selected the AutoProcessing annotation on the mRNA plate is set to RNAPreNormalize. This allows the registration to by-pass the "RNA dilution registration".

  • The lab protocols wizard can now handle plates with pre-normalized RNA.

  • mRNA and cDNA registration flags the parent RNA item in case of a failure.

comment:12 Changed 6 years ago by Nicklas Nordborg

(In [2907]) References #672: RNA concentration normalization wizard

The rest of the library preparation wizards should now work with pre-normalized RNA. Mostly a matter of handling flagging the correct RNA item after a failure.

Manually flagging RNA in the "Create mRNA wizard" is currently done on the pre-normalized RNA item. Since the pre-normalized aliquot is not used up, it would be up to the "Flagged RNA re-processing wizard" to handle if the aliquot or parent RNA item should be used.

comment:13 Changed 6 years ago by Nicklas Nordborg

(In [2908]) References #672: RNA concentration normalization wizard

Fixed the secondary analysis wizard so that they flag the correct RNA item after a failure.

comment:14 Changed 6 years ago by Nicklas Nordborg

(In [2909]) References #672: RNA concentration normalization wizard

Add AutoProcessing and Flag as annotations to RNANormalizedAliquot.

comment:15 Changed 6 years ago by Nicklas Nordborg

(In [2923]) References #672: RNA concentration normalization wizard

Adding a "Toggle all" function to the pre-normalize column so that it is easy switch mode for all items.

comment:16 Changed 6 years ago by Nicklas Nordborg

(In [2930]) References #672: RNA concentration normalization wizard

.n instead of .a in generated labels.

comment:17 Changed 6 years ago by Nicklas Nordborg

Resolution: fixed
Status: assignedclosed
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