Real-time PCR Miner

Sheng Zhao* and Russell D. Fernald

Department of Biological Sciences & Program in Neuroscience

Stanford, California 94305-5020

Frequent Asked Questions:

Q01.     Our Real-time PCR system runs on a Mac. How can we use a PC to use Miner?

Q02.     Why should I always include the cycle numbers as the first column in the data? Are they not just 1, 2, 3...?

Q03.    When I do quantification using the efficiency and CTs computed from Miner, which number should I use, the number for individual sample, for replicates, or for each gene?

Q04.     Can I use the efficiency for each individual sample to do the quantification?

Q05.     Should I always submit my data from only single plates?

Q06.     Why some of my samples are considered as blank by Miner and did not produce any efficiency and CTs?

Q07.     How can I export my data from Bio-Rad MyIQ or iCycler system?

Q08.     How can I export my data from Stratagene MX3000/4000 system?

Q09.     How can I export my data from ABI system?

Q10.     Is there an easy way to tell the data I submitted is pure raw data or baseline subtracted data?

Q11.     How to cite Miner?

Q12.     How to support this website?

Q13.     Why did I get error messages like "Exponential phase fit failed!"?

Q14.     Can I run Miner software locally in my PC?

Q14.     More? coming soon... ...


Q01: Our Real-time PCR system runs on a Mac. How can we use a PC to use Miner?

A01: The program will run on the server and send the results back to you by email, you do not need to run Miner on your own computer.

 

Q02: Why should I always include the cycle numbers as the first column in the data? Why are they not just 1, 2, 3...?

A02: Because in different platforms, they might choose different time for each PCR cycle when recording fluorescence. For example, in ABI systems and Stratagene MX3000/4000, they use 1, 2, 3 for each cycle, while in MyIQ and iCycler from Bio-Rad, they use fractional number for each cycle (eg. 0.58, 1.58, 2.58, etc.)

 

Q03: When I do quantification using the efficiency and CTs computed from Miner, which number should I use, the one for individual sample, for replicates, or for each gene?

A03: In most case, we recommend to use the average efficiency of each gene and the average CTs of each replicates (replicates from the same sample, or "PCR replicates") to do quantification. While when you concerned the tissue or treatment specific inhibitory or activatory effect of PCR reaction for the same gene in your experiment, you might need to use the average efficiency of replicates instead of the average efficiency of each gene. But in this case, you had better use more replicates (6 or more) for each group to get accurate average efficiency from replicates because you have fewer samples to do the average (like only three if you do triplicates). Here is an example in Excel file for how to do the quantification using the average efficiency of each gene and the average CT of replicates.

 

Q04: Can I use the efficiency for each individual sample to do the quantification?

A04: Theoretically yes. Miner does produce the efficiency for each reaction. However, since the exponential phase for each reaction usually contains 6 to 8 cycles (not many points), the estimation for the efficiency from single reaction might be slightly inaccurate although Miner optimizes all the calculation steps. Therefore, we still recommend to use the average efficiency of all samples for each gene (the same primer set) to do the quantification.  In most case, only if you want to know how much do tissue or treatment specific inhibitory and activatory effects of PCR reaction for the same gene exist in your experiment, you might want use the averaged efficiency for replicates instead for each gene to do the quantification computation. Even though, we recommend you use more replicates (more than 5 or 6) instead of only triplicates to run your Real-time PCR experiment.

 

Q05: Should I always submit my data from only single plates?

A05: No. But please make sure you assigned correct title for each samples so that you won't confused when you run a lot of samples. Note, we still think one should at least put the internal control genes together with the interested genes for the same tissue or treatment on the same plate when he run PCR to make sure the normalized data by using reference gene (eg. Actin, G3PDH, 18S rRNA) are comparable.

 

Q06: Why some of my samples are considered as blank by Miner and did not produce any efficiency and CTs?

A06: When the amplification starts very late, these sample even has not finished the exponential phase. These late amplified sample should not be used as the quantification samples and will never accurate because they have not provided enough information yet no matter what kind of method you choose to calculate the efficiency and CTs. When this happened, you can either increase the input concentration in the experiment or use more total cycles for the PCR to allow the reaction curve to at least finish the exponential phase. For the extremely low concentration samples, you might need choose better reverse transcription kit and PCR supper mix to ensure enough amplification.

 

Q07: How can I export my data from Bio-Rad MyIQ or iCycler system?

A07: The MyIQ and iCycler system, they provide "Background subtract data". Right click the mouse on the graph after choose this "Background subtract data" for plotting. Select "Show data" in the popup menu. You will see a excel-like table show up on the top which are the raw fluorescence vs. cycle. Copy and paste the data in table including the cycle numbers to any program which can haddle column data (eg. Excel (<=255 sample if by column), SPSS, and SigmaPlot). After give a suitable title  for each column (in "GeneName_ReplicateName_ ReplicateNumber" format), you can copy the data with the title on the first row into the our website and submit them to Miner for analyses. Note, please do not include the well position information in the submited data (eg. A1, A2, A12...B1, B2...B12...H1, H2...H12). And never use any character except all letters, numbers, and "_"

 

Q08: How can I export my data from Stratagene MX3000/4000 system?

A08: In Stratagene MX3000/4000 system, there are two kinds of fluroscence vs. cycle data you can find, "R multicomponent" and "Rn". "R multicomponent" flurorescence is the pure raw data of the PCR experiment, while "Rn" is the normalized raw data by using ROX internal dye. MX3000/4000 software can export  "Rmulticomponent" (NOT "Rn" or "dRn"!) data to Excel file with a sheet named "Chart Data Excel". But the format of this sheet is quite different from the one Miner needs. We made a "Macro" as below to automate the re-format process. Please select both ROX and FAM for the Amplification Plot (at left bottom corner) before you export the data by using "Chart Data Excel...Format1-Vertical" in "File" menu even you do not use ROX at all (otherwise, you need re-write the Macro below). You can copy and paste the macro code into the exported Excel file's Macro editor and run it. The Macro will generate a new sheet named "Fluo vs. Cycle" with the re-formated data. After give a suitable title  for each column (in "GeneName_ReplicateName_ ReplicateNumber" format), you can copy the data with the title on the first row into the our website and submit them to Miner for analyses. Note, please do not include the well position information in the submited data (eg. A1, A2, A12...B1, B2...B12...H1, H2...H12). And never use any character except all letters, numbers, and "_".

'------------------------------------------------------------------------------------------------------------------------------------------

'Macro for re-formating the exported data from stratagene MX3000/4000 system

'Usually, you only need run once for re-formating the data.

'If you run this macro more than once for the same excel file,

'make sure delete the "Fluo vs. Cycle" sheet or change a name for this sheet first before you re-run it.

'------------------------------------------------------------------------------------------------------------------------------------------

Sub FormatDataForMiner()

      Worksheets(1).Activate

      originalName = Worksheets(1).Name

      Sheets.Add

      Worksheets(1).Activate

      Worksheets(1).Name = "Fluo vs. Cycle" 'Usually, you only need run once for re-formating the data.

                                                                          'If you run this macro more than once for the same excel file,

                                                                          'delete the "Fluo vs. Cycle" sheet or rename it first.

 

      Worksheets(1).Move after:=Sheets(originalName)

      i = 3

      curBlock = Worksheets(originalName).Cells(i, 2).Value

      CycleBlock = curBlock

      If curBlock <> "" Then

            k = 1

            Do

                  Worksheets("Fluo vs. Cycle").Cells(k, 1).Value = curBlock

                  curBlock = Worksheets(originalName).Cells(i + k, 2).Value

                  k = k + 1

            Loop Until curBlock = CycleBlock

            nCycles = k - 2

      End If

      i = i + nCycles + 1

      n = 2

      Do

             curBlock = Worksheets(originalName).Cells(i, 1).Value

             If curBlock = "" Then Exit Do

             Worksheets("Fluo vs. Cycle").Cells(1, n).Value = curBlock

             For k = 1 To nCycles

                    curBlock = Worksheets(originalName).Cells(i + k, 3).Value

                    Worksheets("Fluo vs. Cycle").Cells(k + 1, n).Value = curBlock

             Next

             n = n + 1

             i = i + nCycles + nCycles + 2

      Loop While True

End Sub

'------------------------------------------------------------------------------------------------------------------------------------------

'Macro for re-formating the exported data from stratagene MX3000/4000 system

'Good luck for your Real-time PCR.

'------------------------------------------------------------------------------------------------------------------------------------------

???

Q09: How can I export my data from ABI system?

A09: In ABI system, like 7700, there are two kinds of fluroscence vs. cycle data that you can find, "Rn" and "delta Rn". "Rn" is the reporter signal normalized to the Passive Reference for a given reaction. The delta "Rn" value is the "Rn" value minus the "Rn" value for the No Template Control (kind of baseline). Because Miner will can determine the baseline itself objectively, you should always use the "Rn" data instead of "delta Rn" data.  After choosing "Clipped Data" , the latest version of ABI software (SDS) can exports both the "Rn" and "delta Rn" data to Excel file. For old version of SDS software, one need set both the start cycle and stop cycle of baseline to zero and update the analysis result within SDS software before export the "Clipped Data" to make sure the resulted raw data do not perform any baseline subtraction. After give a suitable title  for each column (in "GeneName_ReplicateName_ ReplicateNumber" format), you can copy the data with the title on the first row into the our website and submit them to Miner for analyses. Note, please do not include the "delta Rn" data and the well position information in the submited data (eg. A1, A2, A12...B1, B2...B12...H1, H2...H12). And never use any character except all letters, numbers, and "_".

 

Q10: Is there an easy way to tell the data I submitted is pure raw data or baseline subtracted data?

A10: Yes. You can have a quick look of the data you exported from the Real-time PCR machine, especially the first 15 row. The number of pure raw data should always larger than the one after baseline subtraction. If you find any negative value or zero, that means you might export the baseline subtracted data. For the machine use the minimal value as baseline, you need choose the data set with larger value.

 

Q11: How to cite Miner?

A11: Sheng Zhao, Russell D. Fernald. Comprehensive algorithm for quantitative real-time polymerase chain reaction. J. Comput. Biol. 2005 Oct;12(8):1045-62. PubMed and PDF

 

Q12: How to support this website?

A12: Thanks for your support. Please read the page for supporting this website.

 

Q13: Why did I get error messages like "Exponential phase fit failed!"?

A13: There are several possible reasons:

1.        Your input data are not in correct format for Miner.

2.        Your input data are not original raw data.

3.        Your data might noisy or not fit the three-parameter simple exponent model.

4.        Your data have unusual efficiency out of the default range.

For reasons 3 and 4, you can try to change the basic options before you run Miner.

 

Q14: Yes, you can download the local command line version of Miner to run in your own PC (Windows only). You can use the option "/?" to get the help for the basic options.

 

Q15: More?

A14: Comming soon ... ..


* Correspondence to:

Sheng Zhao

Lance Kriegsfeld Lab
Neurobiology Research Laboratory
Department of Psychology and
Helen Wills Neuroscience Institute
3210 Tolman Hall
UC Berkeley
Berkeley, CA 94720
Phone: 1-510-643-9899

email: windupzs@gmail.com