Dear all,
I'm currently looking on the expression of specific isoforms from multiple candidate genes.
In order to confirm the expression of these isoforms, my first reaction was to prepare PCR primers enabling to amplify these isoforms either specifically or in duo (two primers for two amplifications of different sizes) and see if the expression of these isoforms changes between my experimental conditions.
However, this method will require further analysis by qPCR and sequencing to confirm the identity of these amplifications and to quantify their changes, which will take a large amount of time.
I was wondering if any faster method could exist to quantitatively analyse specific mRNA isoforms expression. The goal would be to not use omics strategy (as the candidate genes were identified this way) but to use a more targeted and precise approach to look at these specific genes of interest.
Thank you very much for your help.
Dear Matthieu Moncan ,
One alternative method that can be used to quantitatively analyze specific mRNA isoforms expression is RT-qPCR. This method uses reverse transcription to convert RNA into cDNA, and then uses qPCR to quantify the specific cDNA sequences of interest. This method allows for the detection and quantification of specific mRNA isoforms without the need for sequencing. Additionally, it is relatively fast and sensitive, and can be used to compare the expression levels of specific isoforms between different experimental conditions. Another method is using the technology of isoform-specific PCR, where primers are designed to specifically amplify a certain isoform. This method does not require sequencing and is faster than other methods. However, it is important to validate the specificity of the primers with sequencing or other methods.
Thank you Nicolò, my apologies if I didn't express myself correctly but what you are currently suggesting is exactly what I'm currently doing.
As of today, I'm testing the candidate genes with isoform-specific PCR which will then be confirmed by sequencing. Once I have these preliminary results, I will switch to qPCR to assess quantitatively the changes observed by RT-PCR. However, as obtaining primers for specific isoform can sometime be tricky, I also expect to encounter some difficulties when I'm gonna test them at qPCR level to confirm their quality for quantitative results with a standard curve.
It is for these reasons that I wondered if another and faster method would be usable to obtain the same answers in a limited amount of time by reducing the troubleshooting period.
Thanks again for your answer !
Actually, RT-qPCR and RNA-sequencing are the gold standards. Alternatively, Exome Microarray can be used, which is faster but much more expensive and suits more for large-scale studies.
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