Dear Mala, > Hello Dr. Dvinge, > > I have been using HTqPCR 1.8.0 to analyze 96 well qPCR arrays. My > experiment has 2 time point 3hrs, 18hrs and 2 treatments CT, TC and TCATP > (2X3 factorial design). I was able to run the analysis using limmaCtData > and export the output file for each contrast. > > Question 2 > ************************************************************* > Is there an equivalent method like TopTable in limma to extract results > which shows the genes that are changing across the experimental condition > (like F-statistics in ANOVA or F.p.value in limma). We can get this > information using the write.fit() in limma. > Actually no, I never thought about incorporating this. The standard output gives a (slightly modified) version of the results from topTable for each of the coefficients in contrast, as well as decideTests. But not the output from topTableF/topTable with coef=NULL. If this is of interest, I can add it to future versions of HTqPCR? Alternatively, you can just extract the Ct data (using exprs or getCt) and run lmFit/eBayes/topTableF directly. Although this of course won't take the quality of the Ct data into account, i.e. you won't get the columns indicating whether the target and the calibrator samples are OK or Undetermined. Best, \Heidi > Runnning limmaCtData without contrast didn't help. See below > >>DE.limmaNoCtr <- limmaCtData(quan_undtrmnfilter2, design=design, ndups = >> 1, spacing = 1) > >> write.table(qDE.limmaNoCtr, file="qDE_limmafitALL.txt", sep="\t") > ' > Attached file for this: RCode_HTqPCR.txt ; > limmaCtData_output_no_contrast.xls > > ******************************************************************** *** > Thanks for all your help. > > Mala Sinha > Systems Analyst > University of Texas Medical Branch > Galveston, Texas 77573 > masinha at utmb.edu<mailto:masinha at="" utmb.edu=""> > (409) 747 6872 > >

Hi Mala, > Hi, > > Will the results change for the Contrast Analysis from limFit and > limmaCtData? > It should be the same, provided you use the same input data (for example with ndups if you have multiple Ct values for each gene). If you're in doubt, you can always type limmaCtData, without the brackets, to check the code for the function and see how lmFit and eBayes are being used. Best, \Heidi > Thanks. > Mala > > -----Original Message----- > From: Sinha, Mala > Sent: Tuesday, May 29, 2012 9:07 AM > To: 'Heidi Dvinge' > Subject: RE: HTqPCR > > Thanks, will try using limFit with your suggestion. > > Mala > > -----Original Message----- > From: Heidi Dvinge [mailto:heidi at ebi.ac.uk] > Sent: Monday, May 28, 2012 3:38 AM > To: Sinha, Mala > Cc: bioconductor at r-project.org > Subject: Re: HTqPCR > > Dear Mala, > >> Hello Dr. Dvinge, >> >> I have been using HTqPCR 1.8.0 to analyze 96 well qPCR arrays. My >> experiment has 2 time point 3hrs, 18hrs and 2 treatments CT, TC and >> TCATP >> (2X3 factorial design). I was able to run the analysis using >> limmaCtData and export the output file for each contrast. >> >> Question 2 >> ************************************************************* >> Is there an equivalent method like TopTable in limma to extract >> results which shows the genes that are changing across the >> experimental condition (like F-statistics in ANOVA or F.p.value in >> limma). We can get this information using the write.fit() in limma. >> > Actually no, I never thought about incorporating this. The standard output > gives a (slightly modified) version of the results from topTable for each > of the coefficients in contrast, as well as decideTests. But not the > output from topTableF/topTable with coef=NULL. > > If this is of interest, I can add it to future versions of HTqPCR? > > Alternatively, you can just extract the Ct data (using exprs or getCt) and > run lmFit/eBayes/topTableF directly. Although this of course won't take > the quality of the Ct data into account, i.e. you won't get the columns > indicating whether the target and the calibrator samples are OK or > Undetermined. > > Best, > \Heidi > >> Runnning limmaCtData without contrast didn't help. See below >> >>>DE.limmaNoCtr <- limmaCtData(quan_undtrmnfilter2, design=design, ndups >>>= 1, spacing = 1) >> >>> write.table(qDE.limmaNoCtr, file="qDE_limmafitALL.txt", sep="\t") >> ' >> Attached file for this: RCode_HTqPCR.txt ; >> limmaCtData_output_no_contrast.xls >> >> ********************************************************************** >> * >> Thanks for all your help. >> >> Mala Sinha >> Systems Analyst >> University of Texas Medical Branch >> Galveston, Texas 77573 >> masinha at utmb.edu<mailto:masinha at="" utmb.edu=""> >> (409) 747 6872 >> >> > > >