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                                         SuperArray ChIP-qPCR Data Analysis Template
For a detailed discusssion of the calculations used in this file, please see the ChIP-qPCR Primer Assay User Manual.
     Planning the real-time PCR plate layout to match the analysis template before setting up the run can simplify the import process.

     Make a copy of the "ChIP-qPCR Analysis Template" worksheet and reformat it to match your experimental design.
          This worksheet is completely un-protected. Therefore, care must be taken to preserve the cell associations in the formula cells.
          Additional IP fractions can be added by inserting a row within each ChIP Sample ID Section and copying the IP Ab #1 row.
          Delete the italicized Sample Fraction (Template) Rows sections for the optional assays
           Additional samples can be added by inserting enough rows for all Sample Fraction (Template) rows within each Promoter Target
           Site section and copying the Sample Fraction (Template) rows.
           Additional ChIP-qPCR assays can be added by copying a correctly modified Promoter Target Site section and pasting it to the
           bottom of the list.
           Double check the accuracy of ALL cell associations used in the calculation formula cells.
                 Use the "ChIP-qPCR Analysis Example" worksheet as a reference for the original cell associations.

     ChIP-qPCR assay data should be exported from the instrument software in to an Excel spreadsheet.
          The exported data points for each qPCR should include:
                     Reaction well contents label or description
                     Position of the well on the instrument run
                     Cycle threshold value used to determine the Ct values
                     Ct value
                       Peak Tm value from the dissociation analysis1

     Reorganize the exported data rows and columns to match the ChIP-qPCR analysis template structure.
     Copy the appropriate cells from the exported data worksheet and paste them into the modified template worksheet.
          Use the Edit, Paste Special, Value function to preserve the template worksheet formatting.

     Tabular results for three levels of binding site occupancy are returned:
                                  Sample specific: 1) % Input
                                                     2) Assay Site IP Fold Enrichment
                            Relative comparisons: 3) Fold Change in Site Occupancy
     The appropriate results set can be used to generate Excel graphs within the worksheet.


RKB (v. 7/25/07) 6/30/2011
C:\Docstoc\Working\pdf\e923e01a-80ba-46ea-a8f8-17a4f10971d0.xls--Instructions


ChIP sample and experimental quality assessment

         ChIP-qPCR Primer Assay Evaluation
              Confirm that the same Cycle Threshold Value was applied for all Ct values from a single ChIP-qPCR Assay.
               Confirm that all Peak Tm values within a single ChIP-qPCR Assay are within 1 °C of each other1.

         IP Fraction Ct Evaluation
               Check that the Input Ct values are in a good range, generally less than 30.
                     The exact values will vary between different ChIP-qPCR Assays.

               Check that NIS/mock background levels are sufficiently low, generally with “% Input” values less than 0.01%.
                    In the event that the NIS/mock reaction does not generate a Ct value, change the value to 40.

               Check that the Specific IP value is quantitatively above the non-specific background for the sample.
                    Generally, Ct [NIS/mock] – Ct [ChIP] ≥ 1.0, depending on the variation in background level across the samples.

         ChIP- and Antibody-Specific Evaluations

               Check that RNA polymerase II “% Input” is greater than 0.1 % for the Positive Control Proximal HKG Promoter ChIP-qPCR Assay.

               For the Negative Control IGX assay, check that “% Input” for the target specific ChIP fractions is close to or below that of the
               NIS/mock ChIP fraction value.



1
    Not all real-time instrument software exports the peak Tm value. In this case, the T m evaluation should be performed before data export.




RKB (v. 7/25/07) 6/30/2011
         C:\Docstoc\Working\pdf\e923e01a-80ba-46ea-a8f8-17a4f10971d0.xls--ChIP-qPCR Analysis Example 1.0




SuperArray ChIP-qPCR Data Analysis Template                                                    EXAMPLE ONLY
                                                               This worksheet is locked to preserve the cell formula references.

                                                                                               Sample Site Occupancy Results                                                 Differential Occupancy Results
                                                  Copy from qPCR report                             ∆ Ct                     1st ∆∆ Ct                                            2nd ∆∆ Ct
                                                 (all blue values)                                                                      Assay Site IP
       Promoter    ChIP    Sample                                          Peak  Input normalized       % Input    NIS/Mock adjusted Fold Enrichment        Normalized IP        Fold Change in
     Target Site  Sample Fraction                       Plate ThresholdCt   Tm     IP value (∆Ct)      Sites IP'ed IP Ct value (∆∆Ct) above bkgrd (NIS)   Difference (∆∆ Ct)    Site Occupancy
                                                                                                        -∆Ct
(ChIP-qPCR assay)   ID  (Template) Well Description Position for Ct (dRn) (°C) (Ct[IP]-Ct[InputxDF]) ((2 )x100%) (∆Ct[IP]-∆Ct[NIS])        (2-∆∆Ct)     (∆Ct[IP:S2]-∆Ct[IP:S1])       (2-∆∆Ct)                                     Comments
GAPDH                1   Input     GP4(-)Input           A1     0.10 22.99 82.8                    1                                                                                              Sample ID#1 is the experimental control
Proximal             1   Pol 2     GP4(-)Pol             B1     0.10 23.17 82.8         6.82             0.883%           -7.46             176.1
for RNA Pol2         1   IP Ab #1 GP4(-)Ab1              C1     0.10 30.40 83.1        14.05            0.006%            -0.23              1.2
                     1   NIS/mock GP4(-)NIS              D1     0.10 30.63 83.1        14.28             0.005%
ChIP Technical       2   Input     GP4(+)Input           E1     0.10 23.05 83.1
Positive Control     2   Pol 2     GP4(+)Pol             F1     0.10 22.73 82.8         6.32             1.248%           -8.18             290.0                -0.50                  1.4       Ideally 1.0 if no difference in global transcription or IP efficiency
                     2   IP Ab #1 GP4(+)Ab1              G1     0.10 30.15 82.8        13.74            0.007%            -0.76              1.7                 -0.31                  1.2       (Running IP Ab #1 fraction qPCRs is optional on this assay.)
                     2   NIS/mock GP4(+)NIS              H1     0.10 30.91 82.8        14.50             0.004%                                                                 Fold increase in RNA Pol2 binding at the GAPDH proximal promoter in Sample 2.

IGX1A                   1     Input      IGX1A(-)Input          A2      0.10    23.27   77.9
ORF-free                1     Pol 2      IGX1A(-)Pol            B2      0.10    30.52   77.6          13.89             0.007%              -0.23                1.2                                             The specific IP % Inputs should be close to the NIS value.
Intergenic Region       1     IP Ab #1   IGX1A(-)Ab1            C2      0.10    30.02   77.6          13.39             0.009%              -0.73                1.7                                             Specific IP % Input values significantly greater than NIS indicate
                        1     NIS/mock   IGX1A(-)NIS            D2      0.10    30.75   77.6          14.12             0.006%                                                                                   appreciable non-specific gDNA binding by the IP antibody in that fraction.
ChIP Specificity        2     Input      IGX1A(+)Input          E2      0.10    23.45   77.9
Negative Control        2     Pol 2      IGX1A(+)Pol            F2      0.10    31.06   77.6          14.25             0.005%              0.85                 0.6
                        2     IP Ab #1   IGX1A(+)Ab1            G2      0.10    30.98   77.2          14.17             0.005%              0.77                 0.6
                        2     NIS/mock   IGX1A(+)NIS            H2      0.10    30.21   76.9          13.40             0.009%

Assay #1                1     Input      A1(-)Input             A3      0.10    24.22 78.9
                        1     Pol 2      A1(-)Pol               B3      0.10    29.46 79.2            11.88             0.026%             -1.96                 3.9
                        1     IP Ab #1   A1(-)Ab1               C3      0.10    29.85 79.5            12.27             0.020%             -1.57                 3.0
Ab #1 target binding    1     NIS/mock   A1(-)NIS               D3      0.10    31.42 79.5            13.84             0.007%
on promoter site #1     2     Input      A1(+)Input             E3      0.10    24.35 79.2
                        2     Pol 2      A1(+)Pol               F3      0.10    28.14 79.2            10.43             0.072%             -3.34                10.1               -1.45              2.7        (Running IP Pol 2 fraction qPCRs is optional on this assay.)
                        2     IP Ab #1   A1(+)Ab1               G3      0.10    26.74 78.9             9.03             0.191%             -4.74                26.7               -3.24              9.4
                        2     NIS/mock   A1(+)NIS               H3      0.10    31.48 78.9            13.77             0.007%                                                                 Fold increase in [target] binding at [this site] in Sample 2.

Assay #2                1     Input      A2(-)Input             A4      0.10    25.32 77.9
                        1     Pol 2      A2(-)Pol               B4      0.10    30.56 78.2            11.88             0.026%             -1.76                 3.4
                        1     IP Ab #1   A2(-)Ab1               C4      0.10    30.95 78.5            12.27             0.020%             -1.37                 2.6
Ab #1 target binding    1     NIS/mock   A2(-)NIS               D4      0.10    32.32 78.5            13.64             0.008%
on promoter site #2     2     Input      A2(+)Input             E4      0.10    25.45 78.2
                        2     Pol 2      A2(+)Pol               F4      0.10    29.84 78.2            11.03             0.048%             -2.94                7.7                -0.85              1.8        (Running IP Pol 2 fraction qPCRs is optional on this assay.)
                        2     IP Ab #1   A2(+)Ab1               G4      0.10    27.54 77.9             8.73             0.235%             -5.24                37.8               -3.54              11.6
                        2     NIS/mock   A2(+)NIS               H4      0.10    32.78 77.9            13.97             0.006%                                                                 Fold increase in [target] binding at [this site] in Sample 2.

                              1. In this example the Input fraction was 1% of the chromatin used in the IP fractions. Therefore the dilution factor (DF) is 100 or 6.644 cycles.




         RKB (v. 7/25/07) 6/30/2011
         C:\Docstoc\Working\pdf\e923e01a-80ba-46ea-a8f8-17a4f10971d0.xls--ChIP-qPCR Analysis Template 1.0


                                                                                                                     SuperArray ChIP-qPCR Data Analysis Template
1
 In this worksheet the default Input fraction is 1% which is a dilution factor (DF) of 100 or 6.644 cycles (i.e. log 2 of 100).
2
 Ideally, this value should be 1.0 if there is no difference in global transcription or IP efficiency.
Note: A 1.0 Fold Change in Site Occupancy is no change.
                                                                                                                   Sample Site Occupancy Results                          Differential Occupancy Results
                                                       Copy from qPCR Report                                      ∆ Ct                    1st ∆∆ Ct                                   2nd ∆∆ Ct
                                                       (use Edit/PasteSpecial/Values)                                                                      Assay Site IP
      Promoter          ChIP  Sample                                                         Peak   Input normalized       % Input    NIS/Mock adjusted Fold Enrichment        Normalized IP       Fold Change in
     Target Site       Sample Fraction                              ThresholdCt
                                                                  Plate                       Tm      IP value (∆Ct)      Sites IP'ed IP Ct value (∆∆Ct) above bkgrnd (NIS) Difference (∆∆ Ct)     Site Occupancy
                                                                                                                           -∆Ct
(ChIP-qPCR Assay)        ID     (Template) Well Description Position for Ct (dRn)            (°C) (Ct[IP]-Ct[InputxDF]) ((2 )x100%) (∆Ct[IP]-∆Ct[NIS])        (2-∆∆Ct)     (∆Ct[IP:S2]-∆Ct[IP:S1])      (2-∆∆Ct)                                       Comments
GAPDH                    1      Input                                                                                                                                                                               ChIP Sample ID #1 is the control sample for the experiment.
Proximal                 1      Pol 2                                                                     6.64              1.000%         0.00               1.0
for RNA Pol2             1      IP Ab #1                                                                  6.64              1.000%         0.00               1.0
                         1      NIS/mock                                                                  6.64              1.000%
ChIP Technical           2      Input
Positive Control         2      Pol 2                                                                     6.64              1.000%         0.00               1.0                 0.00                 1.0          Fold increase in RNA Pol II binding at HKG Proximal Promoter in Sample 2.2
                         2      IP Ab #1                                                                  6.64              1.000%         0.00               1.0                 0.00                 1.0          (Optional)
                         2      NIS/mock                                                                  6.64              1.000%

IGX1A                     1     Input
ORF-free                  1     Pol 2                                                                      6.64             1.000%         0.00               1.0                                                   The Assay Specific IP Fold Enrichments should be close to the NIS/mock value.
Intergenic Region         1     IP Ab #1                                                                   6.64             1.000%         0.00               1.0                                                   Specific IP Fold Enrichments significantly greater than the NIS/mock indicate
                          1     NIS/mock                                                                   6.64             1.000%                                                                                  appreciable non-specific gDNA binding by the IP antibody in that fraction.
ChIP Specificity          2     Input
Negative Control          2     Pol 2                                                                      6.64             1.000%         0.00               1.0
                          2     IP Ab #1                                                                   6.64             1.000%         0.00               1.0
                          2     NIS/mock                                                                   6.64             1.000%

Assay #1                 1      Input
                         1      Pol 2                                                                     6.64              1.000%         0.00               1.0
                         1      IP Ab #1                                                                  6.64              1.000%         0.00               1.0
Ab #1 target binding     1      NIS/mock                                                                  6.64              1.000%
on promoter site #1      2      Input
                         2      Pol 2                                                                     6.64              1.000%         0.00               1.0                 0.00                 1.0          (Optional)
                         2      IP Ab #1                                                                  6.64              1.000%         0.00               1.0                 0.00                 1.0          Fold increase in [target] binding at [this site] in Sample 2.
                         2      NIS/mock                                                                  6.64              1.000%

Assay #2                 1      Input
                         1      Pol 2                                                                     6.64              1.000%         0.00               1.0
                         1      IP Ab #1                                                                  6.64              1.000%         0.00               1.0
Ab #1 target binding     1      NIS/mock                                                                  6.64              1.000%
on promoter site #2      2      Input
                         2      Pol 2                                                                     6.64              1.000%         0.00               1.0                 0.00                 1.0          (Optional)
                         2      IP Ab #1                                                                  6.64              1.000%         0.00               1.0                 0.00                 1.0          Fold increase in [target] binding at [this site] in Sample 2.
                         2      NIS/mock                                                                  6.64              1.000%




         RKB (v. 7/25/07) 6/30/2011

				
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