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Consistancy in qPCR data


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5 replies to this topic

#1 shyamadi

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Posted 31 May 2011 - 12:12 AM

Hello everybody,
Though I am very new to this technique, I am performing qPCR with full trust on me. I don't know what is wrong with me as I am having ridiculous problem of data inconsistency despite of every defined and alike conditions. My sample is same, my primer is same, everything thing is well so far my knowledge, but why sometimes I am getting higher signal and sometimes getting extremely lower signal for the same gene in same conditions. My data are comically very variable. I don't know what to do, where is the problem?? Please anybody help me!!! any help for this problem will be greatly appreciated by this amateur learner.

Sam

#2 Lapsang

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Posted 31 May 2011 - 08:05 AM

Can you give a little bit of extra info to help us figure out possible causes?

Is the qPCR looking at gene expression (e.g. you are using it on cDNA made from mRNA for a gene of interest) or is it after a ChIP or DIP, to look at transcription factor binding or methylation at your gene of interest?

When you say it is variable, how much does it typically vary by? Can you give any example data (e.g. Ct values)? Do you get variation in any controls you use (if you have any)?

A rough guide of your experiments up until the qPCR will be useful. example: Cells --> RNA isolation by trizol --> cDNA synthesis --> qPCR

#3 shyamadi

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Posted 01 June 2011 - 01:15 AM

Can you give a little bit of extra info to help us figure out possible causes?

Is the qPCR looking at gene expression (e.g. you are using it on cDNA made from mRNA for a gene of interest) or is it after a ChIP or DIP, to look at transcription factor binding or methylation at your gene of interest?

When you say it is variable, how much does it typically vary by? Can you give any example data (e.g. Ct values)? Do you get variation in any controls you use (if you have any)?

A rough guide of your experiments up until the qPCR will be useful. example: Cells --> RNA isolation by trizol --> cDNA synthesis --> qPCR


Dear Sir,
Thank you very much for your kindness.
Yes I am trying to look the gene expressions using cDNA. I am using 1:100 cDNA dilution and my primers conc. are 20 pmole. My total reaction volume is 20ul. After qPCR run, I am getting Ct value less than 15, but I have heard somewhere it should be in between 15 to 25. I really dont have any idea. I have hereby, attached my data sheet for your review.
My experiment outline is as follow:
1) I treated HaCaTs cells with my compound of interest.
2) Isolated mRNA using Trizol Method and quantified.
3) cDNA was synthesized using 1 ug of RNA and then diluted x100 for qPCR.
I have treated my cell with compound in different time intervals and tried to evaluate some gene expression level as mentioned in my data sheet.

Thanking you very much and expecting your kindness for my problem.

Attached Files



#4 shyamadi

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Posted 01 June 2011 - 02:45 AM

I am using delta delta Ct method and looking for expression of genes normalized with GAPDH and hence I am getting relative concentration which is mentioned in my data sheet.

Thanking you !!
:D

#5 Lapsang

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Posted 01 June 2011 - 01:04 PM

OK, thanks for sharing your data.

First I would think of basic problems (sorry if they seem obvious, but you said that you are new to qPCR).

Did you take care to avoid RNA degradation? (e.g. by using filtered pipette tips) If you run your RNA on a gel do you see distinct bands or do you see a smear? Degraded RNA can give odd results in when you try to use it for qPCR.

Do the primers you use span an intron? If your primers are not intron spanning they will also amplify any genomic DNA contaminating your samples which has not been degraded by DNase, which could explain you getting lower Ct values than you expected. Did you do a control cDNA synthesis (reaction with everything except reverse transcriptase) and also run this on the qPCR?

Edited by Lapsang, 01 June 2011 - 01:04 PM.


#6 shyamadi

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Posted 01 June 2011 - 08:46 PM

OK, thanks for sharing your data.

First I would think of basic problems (sorry if they seem obvious, but you said that you are new to qPCR).

Did you take care to avoid RNA degradation? (e.g. by using filtered pipette tips) If you run your RNA on a gel do you see distinct bands or do you see a smear? Degraded RNA can give odd results in when you try to use it for qPCR.

Do the primers you use span an intron? If your primers are not intron spanning they will also amplify any genomic DNA contaminating your samples which has not been degraded by DNase, which could explain you getting lower Ct values than you expected. Did you do a control cDNA synthesis (reaction with everything except reverse transcriptase) and also run this on the qPCR?


Thank you very much Sir for thorough consideration of my problems. I think my RNA was fairly good when I visualized. It was neither sharp band nor smeared but a bit of fuzzy. Regarding my primers, I designed using gene specific mRNAs in NCBI and the sizes are less than 200 bps. I think I must have spanned the intron sequence, however I am not sure. I would find some solid informations and will let you know. The idea about the control cDNA synthesis wasn't, of course in my mind. I am going to repeat my experiment caring all basic steps as you have mentioned and will let you know.
Thank you very much for your brilliant idea and hoping to get more and more.

Sam







































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