• Generally speaking, you want to design primers to fit most of the following criteria: Unique (i.e. no off targeting, no primer dimer) - you can test this with a blast search or UCSC’s in silico PCR tool. Primer melting temperature has compatibility with the polymerase you will use (Typically around 60-63C). Also make sure the melting temperature of the primers are not too different. The amplicon spans exon-exon junctions for specificity purpose. Amplicon is a long enough PCR product that can be properly amplified. Some also say that high CG content towards the 3’ end of primer yields higher specificity.

2. Most eRNAs are inherently short and unstable, therefore they are hard to detect by most steady-state RNA assessment techniques. The best way to identify eRNAs is to use a nascent RNA sequencing technique such as PRO-seq or GRO-seq. These techniques successfully capture RNAs engaged with transcriptionally active RNA polymerase II (before the transcripts are degraded by the exosome). I’m sure there are other creative ways of detecting eRNAs, but these are two well-proven methods. Not to mention the assessments you'll have to do to be able to properly call the region where you detect the "eRNA" an enhancer (i.e. ChIP-seq - hPTMs, RNAPII, p300, and accessibility assessments- ATAC-seq, DNase-seq).

I've done this successfully by normal design of short RT-qPCR primers (~80 nt size product), but you absolutely HAVE to use DNase when preparing your RNA, and you HAVE to have a -RT control sample, so you ensure you aren't amplifying chromosomal DNA instead of your cDNA. Obviously, you have to prime your cDNA with random hexamers and not poly-T.

these links are useful

https://www.sciencedirect.com/science/article/.../S167202291730076...

https://www.systembio.com/.../Regulatory-RNA_Manual_WEB1-1.p...

regards