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Hi everyone,
Actually, i am working on gene expression project and aimed to use RT-PCR and NGS methods. The NGS method work well BUT the PCR product for RT-PCR method was poor.
I designed the primers using primer-BLAST and then run PCR to check the presence and absence of the product. I run the gel but there was no products/no amplifications. I tried other primers but still no products. My target sequences have a lots of low complexity regions, and based on what i read the low complexity regions consume the PCR product and caused the failure PCR (no products). So I concentrated on the NGS method only instead of using RT-PCR. My question is ..is this enough to justify the reason of excluded the RT-PCR method cause of the low complexity regions which is led to the failure PCR or i should try other primers until its work ?? Or there is another possible reasons to get this failure PCR?
Thanks
Actually, i am working on gene expression project and aimed to use RT-PCR and NGS methods. The NGS method work well BUT the PCR product for RT-PCR method was poor.
I designed the primers using primer-BLAST and then run PCR to check the presence and absence of the product. I run the gel but there was no products/no amplifications. I tried other primers but still no products. My target sequences have a lots of low complexity regions, and based on what i read the low complexity regions consume the PCR product and caused the failure PCR (no products). So I concentrated on the NGS method only instead of using RT-PCR. My question is ..is this enough to justify the reason of excluded the RT-PCR method cause of the low complexity regions which is led to the failure PCR or i should try other primers until its work ?? Or there is another possible reasons to get this failure PCR?
Thanks