Biogazelle qBasePlus questions & answers

qBasePlus showed the 'NaN' at the Reference target stability for both reference genes I chose to use. And while checking Analysis tab to delete the genes with 'NaN' mark, I found most of samples have 'NaN' result for all primers, including two reference genes and three target genes. 2/3 of samples are from the former experiment and remains are from the current expt. but process was all the same. This is the first time for us to encounter this problem.

Just in case, I attachd the protocol I followed for LinReg and qBase Plus, and if there is any other information I have to deliever to solve this issue, please let me know.:

qPCR Analysis: qBASE and LinReg

1. Preparation of Excel sheets for qBASE
   Open the run (CFX file) using Bio-Rad CFX Manager
   a) Prepare an excel sheet first
   b) In CFX file, go to quantification data, copy the whole thing and paste it into excel sheet
   c) Delete Cq mean and Cq standard deviation columns. Leave Column A empty
   d) Then highlight columns. Click on Daten, then Filter
   e) Click on arrow Cq – Nach Grösse sortieren (aufsteigend)
   f) Click on sample- Nach Farbe sortieren – Benutzer definiertes sortieren – sortieren nach – select sample
   g) Click on target- Nach Farbe sortieren – Benutzer definiertes sortieren – sortieren nach – select target
   h) Open the Excel file named “Code for qBase excel file CORRECTED”
   i) Copy paste the formula between orange borders to the first three rows of samples (triplicates)
   a. = IF(I4="Repeat !","Repeat !", IF(AND( ABS(H3-H2)> 0.5,ABS(H4-H2) > 0.5), "Remove",""))
   b. = IF(I4="Repeat !","Repeat !", IF(AND( ABS(H3-H2)> 0.5,ABS(H4-H3) > 0.5), "Remove",""))
   c. = IF(AND( ABS(H3-H4)> 0.5,ABS(H4-H2) > 0.5, ABS(H3-H2)> 0.5), "Repeat !",IF(AND( ABS(H4-H3)> 0.5,ABS(H4-H2) > 0.5),"Remove",""))
   j) Then highlight the three rows containing formulas and paste it to remaining samples (triplicates)
   k) Save a non-deleted version of the file as ‘qBASE non-deleted + information’
   l) Delete the rows that have been labelled with ‘Remove’
   m) Delete rows with ‘Zeile löschen’ and take notes of the ones you should repeat (if you have any)
   n) Delete repeat samples with ‘probe wiederholen’
   o) Create separate excel sheets for housekeeping genes and gene of interest.
   p) Save the excel sheets in a gene-specific manner (for ex., one Excel file for ACTB, another one for GAPDH, etc) in a single folder. Sample names in both excel sheets should be same. There shouldn’t be discrepancy in the sample names
   Attention!
   If you need to repeat one or more samples, copy the results from the repetition into the original file and save it. This is the file you will need to run the qBASE software.
   By preparing the Excel files’ first, you will be able to know if you need to repeat samples before running the whole analysis on LinReg and qBASE softwares.

2. Running LinReg to get amplification efficiencies
   Export data from CFX software:

• Open the CFX file containing the results (.prcd)
• Go to the “Quantification Data” tab
• Choose RFU
• Go to “quantification data” and choose RFU
• Settings  “Analyze mode”  “Target
• Settings  no baseline substraction
• Select the whole table by clickling on the top left corner (next to ‘Cycle’)
• Right-click on the same spot and select “Export to Excel…”
• Save it on the folder you wish
• Close the CFX file and do not save any changes
  In Excel:
• Delete the first column (it should be empty anyway)
• Delete NTCs and samples that you don’t want to calculate the efficiency
• Replace failing data with new one (If you repeated any samples, you have to replace the old data with the new data from the repetition into the LinReg file. Quick tip: change the color of these special cases ;)
• Add a new row
• Write “Well” in A1
• Name the wells after your primer pair used, eg. Ubc-1, Ubc-2 (note: let a space or a hyphen in between name and number!)
• Leave the Excel open!
  Import data to LinReg:
• Open LinReg, now you see an input window
• Choose “DNA binding dye” for SYBR green, “Biorad iCycler”, “ss cDNA” [I think so at least, because the other option is not cDNA?], “no baseline substraction”
• Choose the right file and the right sheet (LinReg always shows the sheet first that is open in Excel)
• Choose the coordinates of the table you want to import (“A – xxx” and “1-“)
• Press “OK”
• Press the red button! Now LinReg determines the baselines
• NOTE: if an error comes about noisy, adjust the threshold on the left side, until the samples (red dots on the bottom of the software) look homogeneous height-wise
  Export data to Excel:
• After you have inspected all samples and you have excluded some, press “recalculate W-o-L”
• Now you can export your data to excel: “File”  “Save to Excel”  choose “compact” or “compact and complete”
• Use the mean efficiency for calculation of relative expression data (Q-values)
• Check how many samples have been excluded from the calculation of the mean efficiency, if there are a considerable amount, it might be that either your pcr run was very irregular or one of your experimental conditions influenced the efficiencies (e.g. if you have very different concentrations). In latter case, run LinReg for your groups separately and compare the resulting mean efficiencies. If they are not the same, use each of them separately and do not use the mean efficiency calculated from all the samples

3. Running qBASE
   a) Start the SEH UTN Manager first
   b) Right-click on ‘qBASE’ and activate it
   c) Click on qBASE logo
   d) Then click on install, if necessary
   e) Then browse- 10$, Neurobiochemisches labor, Linreg and qBase and select 000CF6AA47DF-2.3_premium.lic. Finish. Then ok.
   f) Add new project (File->New->Project). Name it.
   g) Click on experiment. Add new experiment (File->New->Experiment). Name it.
   h) Then right click on Runs. Import runs. Browse the file. Select File type- CFX, Finish.
   i) Check whether all samples are uploaded by clicking on Sample
   j) Then click on Targets, transfer Targets of interests (housekeeping genes) to reference targets by right clicking on the gene- Set target type – Reference target
   k) Click on settings, click on calculation parameters, click on target specific amplification efficiencies
   l) Click on intermediate results, then click on amplification efficiencies. Then write in the PCR efficiencies for each gene calculated by Linreg.
   m) Click on Quality control, then on Reference target stability. If everything is green then nothing to worry. If it doesn’t have numbers and only NaN then there are some samples which did not work with housekeeping genes, these samples have to be deleted. These can be checked in Analysis, then on Result table. Delete samples which have NaN in housekeeping genes columns.
   n) If its not green, then click on settings, quality control settings, then change Reference target stability- geNorm M value to 0.7 (you can increase up to 1), increase Reference target stability- co-efficient of variation to 0.3 (up to 0.5).
   o) Out of 6 housekeeping genes, select the three or four most stable house keeping genes (green in quality control)
   p) Click on Analysis, then on Result table. Then right click on table, copy and paste it in excel. Use gene CNRQ values not SE (CNRQ) for further analysis. These gene CNRQ values are the normalised mRNA levels for that gene.