Question: Inconsistencies between RNA Seq and PCR
1
gravatar for Bnf83
2.2 years ago by
Bnf83110
Bnf83110 wrote:

Hi guys, I have a question concerning RNA_Seq and PCR analysis results. We performed an experiment in which we interfered with the expression of one gene of interest in order to change the effect on the functionally correlated genes. We performed the experiment using 2 replicates: 2 for treatment (interference) and 2 for control (not interfered gene). Through the PCR we verified that the gene was interfered properly and hence not expressed and this resulted with respect to both controls. Then we performed the RNA_Seq analysis on the full transcriptome but surprisingly there is not a statistically significant difference between the expression of the gene of interest between the treatment and the control although the cell lines were not changed between PCR and RNA_Seq, i.e. the samples were the same. We expected a confirmation of the PCR experiment.

Can anyone help me to explain such a strange behaviour between the two experiments?

Kind regards

B.

pcr interference rna seq • 1.2k views
ADD COMMENTlink modified 2.2 years ago by WouterDeCoster29k • written 2.2 years ago by Bnf83110

There are so many potential sources of error in both experiments that it is difficult to know where to start with an answer. How many house keeping genes did you use to normalize your qPCR? What primers did you use?

To be honest, this isn't something someone without a deeper understanding of your project could help you with. 2 replicates for RNA-Seq may not be enough to detect differences in lowly-expressed genes...

ADD REPLYlink written 2.2 years ago by John12k

@John is right, there are many potential errors in the experiment.

How did you 'interfere' with gene expression? Was it a transient siRNA? A stable knockdown? A knockout? Stable shRNAs can be silenced over time. Knockouts (depending on the target site) can still be transcribed but yield non-functional mRNA.

What degree of downregulation did you get by qPCR? 20%? 50%? 90%?

ADD REPLYlink written 2.2 years ago by jotan1.2k

The interference was done with transient siRNA by a technician in my lab. She observed ~2 fold decrease with the control (scramble).

ADD REPLYlink written 2.2 years ago by Bnf83110

Can you tell me how you know that:

Knockouts (depending on the target site) can still be transcribed but yield non-functional mRNA.

I am trying to find a paper but I can't:(

ADD REPLYlink written 5 months ago by kjdaescu0

If you introduce a frameshift/nonsense mutation your RNA can still be transcribed but lead to nonsense mediated mRNA decay (NMD)

ADD REPLYlink written 5 months ago by WouterDeCoster29k

How did you do the RNA-Seq analysis? Do you have any numbers and units of measure?

ADD REPLYlink written 2.2 years ago by karl.stamm3.2k

Concerning the RNA Seq analysis, I followed the "best practice guidelines". Specifically (and briefly), I performed the alignment, the merge of the lines, I removed the duplicated, the sorting and I performed the reads count using "htseq-count -t exon -i gene_id" . Then I used the reads counts to perform the differential expression analysis using DESeq.

ADD REPLYlink written 2.2 years ago by Bnf83110

Remove duplicates is for calling SNPs in genomic data. In quantitative RNA-Seq you are removing more signal. Most read-duplicate software I know of looks at just the start and end coordinate, and assumes replications come from amplification. The best practices are for genomic variant calling. I think you want to count reads including duplicates to get everything that was sequenced. Your alignment parameters will also influence how many reads are seen at the gene, but hopefully for both samples in the same way.

ADD REPLYlink written 2.2 years ago by karl.stamm3.2k
0
gravatar for WouterDeCoster
2.2 years ago by
Belgium
WouterDeCoster29k wrote:

Where in the gene did your qPCR primers and probe anneal? Is it possible that there are multiple transcripts, of which indeed one is influenced by your treatment and the rest aren't?

ADD COMMENTlink written 2.2 years ago by WouterDeCoster29k

I think this might be the case. I have to add that during the RNA Seq analysis I mapped the reads on the exons and then I performed the counting. I did not considered splicing variants and other stuffs just because we are interested to see if after the interference there's an effect on gen expression independently from the transcript variants.

ADD REPLYlink written 2.2 years ago by Bnf83110

You could consider to only count the reads for the exon you used for the qPCR assay.

ADD REPLYlink written 2.2 years ago by WouterDeCoster29k
Please log in to add an answer.

Help
Access

Use of this site constitutes acceptance of our User Agreement and Privacy Policy.
Powered by Biostar version 2.3.0
Traffic: 596 users visited in the last hour