Very interesting question. I suppose it is possible. You probably will need to make a Rt PCR to get the cDNA. So by the end you would not make it any easier.

If you only want to know if there is a mutation there are simple methods that do not need sequencing. Now if you want to know the exact nature of mutation sequencing cannot be avoided.

If you only want to detect the expression of the gene, then you can go for RT-PCR. but as you said the mutation in DNA causes reduced mRNA expression, then it is better to go for RT-qPCR.

Hi Abouzar ,

in my opinion, either detecting the mutation on the genome (DNA) level or detecting the reduced transcription level on the transcript (RNA) level is a valid experiment. My favorite solution would be to do both and showing that there is a direct correlation between clones which have the mutation and clones which don't have it as a proof.

I agree with Tomas, that it doesn't get easier by detecting the lower transcription levels. With other words, why is the mutation detection such a problem for you? For example, have a look on the HiDi DNA polymerase from myPOLS Biotec. You can simply detect any mutation by using an allele-specific primer and do PCR with them...

Dear Abouzar,

You've did not mentioned where the mutation is located, you've wrote just "DNA" - my question is whether it is a coding or non coding region, including promotor(s), intron(s), UTRs.

If you can prove the hypothesis, for a certain gene, mutation X in DNA in exact locus determines mRNA expression decrease (another question if it is low mRNA quantity or it is not functional because of the mutation which trouble mRNA folding and protein synthesis), then you may use them equally. But, I assume you would need to prove it in a cell/animal model or test on a large number of patients.

Also, I would agree with Ramon - there are PCR/qPCR based solutions for mutation detection, e.g. TaqMan based for SNPs. Selecting between DNA or RNA, I would rely more on DNA, just because RNA quantification apply indirect calculations, depends on a corresponding reference gene etc. Anyway, sequencing would give you hints to prove same nucleotide changes in DNA sequence and it's corresponding mRNA, the idea Tomas had in mind.

And again, it would depend on the nature of the mutation - how many nucleotides are affected, lack of inversions, large indels etc.

Good luck!!!

Firstly, not always a mutation leads to reduced mRNA expression. As a gold standard I would always start with screening my mutations in DNA, followed by pointing out the exact location and nature of the mutation. Then only any further experimentation on RNA level would provide you some relevant information. For example if your mutation affects the mRNA levels in comparison to healthy controls, then you can assume the mutation to be pathogenic and might be disease causing. I recommend this rational approach. Also, detection on transcriptional level is always tricky in comparison to genome (DNA) level. Amplifying DNA should not be a problem. Ampli-taq gold polymerase is great for difficult target regions. If you want to screen longer DNA fragment (>5kb) then opt for Takara Clontech Long Range PCR kit. It is very robust and works very efficiently.

Technically it is possible. However there are a lot of factors that determine which method (DNA mutation detection or expression level measurement) to use.

If it is well established that a specific mutation in a DNA causes reduction in expression of a specific mRNA that leads to symptoms of the disease under question, I suppose it would be better to go for mutation detection, because mutation detection is simpler and more direct test than mRNA quantification.

If the relationship between the mutation and gene expression is not well established but the effect of reduced gene expression is sure to cause the disease, mRNA quantification is the better method. The mRNA quantification is preferable to detect the gene expression alteration for whatever reasons.

Good luck

I agree with all above.Checking mRNA expression of mutated gene and detection of mutation in DNA could be mutually inclusive.