Artefact with Digital Gene expression? unable to confirm downregulation.

Flamant · 11-23-2010, 05:00 AM

I am puzzled by discrepancies between DGE results obtained with SOLID and Q-RT-PCR confirmation in one experiment where we looked at the response of a cell line to one drug, after 24 hours: whereas we can confirm in all tested cases mRNA upregulation, we were unable to confirm downregulation: In each case (10/10) DGE indicates significant expression level (>500 reads) and clear downregulation, whereas no change was observed by Q-RT-PCR, on the same samples. PCR primers were in 3'Unt regions.

We made attempts trying to understand this discrepancy:
- using oligodT or random primers for Q-RT-PCR has no influence.
- Q-RT-PCR does not reveal downregulation at later time points (48h).
- biological repeats will be runned soon.

Did anyone already observed the same problem? Any possibility that DGE can go wrong?

pmiguel · 11-23-2010, 12:36 PM

Maybe your sequencing tags are more gene-specific than your qRT-PCR primers? That is, are you sure your qPCR primers amplify a transcript from only one locus? Maybe there is another transcribed gene with the same 3' Unt priming sites. 3' untranslated regions sometimes have repetitive transposable elements embedded in them.

What type of sequence tags were these? Single or paired end?

--
Phillip

Flamant · 11-25-2010, 12:54 AM

This is a possibility. These were single tags. Yes, the possibility exists that sometimes we are looking at different mRNA with the two techniques (although we used Blast to ascertain that PCR primers were specific and use only reads without mismatches in SAGE) but what is puzzling is that this seems to be a systematic problem: only for downregulated genes, and for 5 out of 5

Thank you

pmiguel · 11-29-2010, 06:58 AM

Another possibility: your qPCR kit is contaminated. How do your negative controls look?

--
Phillip

Flamant · 11-29-2010, 07:53 AM

We always verified contamintation so this is not the explanation. Thank you for helping

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11-23-2010, 05:00 AM	#1
Flamant Junior Member Location: France Join Date: Nov 2010 Posts: 3	Artefact with Digital Gene expression? unable to confirm downregulation. I am puzzled by discrepancies between DGE results obtained with SOLID and Q-RT-PCR confirmation in one experiment where we looked at the response of a cell line to one drug, after 24 hours: whereas we can confirm in all tested cases mRNA upregulation, we were unable to confirm downregulation: In each case (10/10) DGE indicates significant expression level (>500 reads) and clear downregulation, whereas no change was observed by Q-RT-PCR, on the same samples. PCR primers were in 3'Unt regions. We made attempts trying to understand this discrepancy: - using oligodT or random primers for Q-RT-PCR has no influence. - Q-RT-PCR does not reveal downregulation at later time points (48h). - biological repeats will be runned soon. Did anyone already observed the same problem? Any possibility that DGE can go wrong?

11-25-2010, 12:54 AM	#3
Flamant Junior Member Location: France Join Date: Nov 2010 Posts: 3	May be This is a possibility. These were single tags. Yes, the possibility exists that sometimes we are looking at different mRNA with the two techniques (although we used Blast to ascertain that PCR primers were specific and use only reads without mismatches in SAGE) but what is puzzling is that this seems to be a systematic problem: only for downregulated genes, and for 5 out of 5 Thank you

11-23-2010, 12:36 PM	#2
pmiguel Senior Member Location: Purdue University, West Lafayette, Indiana Join Date: Aug 2008 Posts: 2,317	Maybe your sequencing tags are more gene-specific than your qRT-PCR primers? That is, are you sure your qPCR primers amplify a transcript from only one locus? Maybe there is another transcribed gene with the same 3' Unt priming sites. 3' untranslated regions sometimes have repetitive transposable elements embedded in them. What type of sequence tags were these? Single or paired end? -- Phillip

11-29-2010, 06:58 AM	#4
pmiguel Senior Member Location: Purdue University, West Lafayette, Indiana Join Date: Aug 2008 Posts: 2,317	Another possibility: your qPCR kit is contaminated. How do your negative controls look? -- Phillip

11-29-2010, 07:53 AM	#5
Flamant Junior Member Location: France Join Date: Nov 2010 Posts: 3	We always verified contamintation so this is not the explanation. Thank you for helping