Hi Mohammad Alshboul

General steps: RNA/miRNA extraction-> cDNA synthesis-> qRT-PCR (SYbr/Taqman)-> miRNA primers.

->RNA in plasma and serum is normally fragmented RNA or small RNA, mainly miRNA, and is either bound to proteins or contained inside extracellular vesicles. You can look Norgen exosomal RNA purification mini kit. Even Qiagen and Thermo have miRNA extraction kits, whichever you find cost-effective, you can go for it.

Next, you can go with Qiagen miScript RT kit II. The kit basically converts all miRNA present in particular RNA samples into miRNA specific cDNA. That cDNA can be used directly with SYBr based qRT-PCR. So, here if you get different miRNA primers of your interest, you can analyze them with that miRNA specific cDNA with SYBr based qRT-PCR.

NOTE: I. Here using that Qiagen cDNA kit (Qiagen miScript RT kit II) only, you can go for SYBr. Since many including me found that the Thermo miRNA advanced cDNA kit generally does not work for SYBr, so you have to get the Taqman probe.

II. Prefer pre-made miRNA primers from Qiagen, which comes in LNA tech-based and ready to use.

Hope this may help you.

Regards

Saurabh

Hi Mohammad Alshboul

General steps: RNA/miRNA extraction-> cDNA synthesis-> qRT-PCR (SYbr/Taqman)-> miRNA primers.

->RNA in plasma and serum is normally fragmented RNA or small RNA, mainly miRNA, and is either bound to proteins or contained inside extracellular vesicles. You can look Norgen exosomal RNA purification mini kit. Even Qiagen and Thermo have miRNA extraction kits, whichever you find cost-effective, you can go for it.

Next, you can go with Qiagen miScript RT kit II. The kit basically converts all miRNA present in particular RNA samples into miRNA specific cDNA. That cDNA can be used directly with SYBr based qRT-PCR. So, here if you get different miRNA primers of your interest, you can analyze them with that miRNA specific cDNA with SYBr based qRT-PCR.

NOTE: I. Here using that Qiagen cDNA kit (Qiagen miScript RT kit II) only, you can go for SYBr. Since many including me found that the Thermo miRNA advanced cDNA kit generally does not work for SYBr, so you have to get the Taqman probe.

II. Prefer pre-made miRNA primers from Qiagen, which comes in LNA tech-based and ready to use.

Hope this may help you.

Regards

Saurabh

Agree with Saurabh Mandal. The first option is the best solution for your concern, I guess.

Hi Mohammad Alshboul, you can use NucleoSpin miRNA Plasma kit for isolating miRNA. Subsequently, Poly-A tailing can be performed using a kit from Invitrogen Thermofisher Scientific followed by PrimeScript 1st strand cDNA Synthesis from Takara Bio Inc. For more specific details of the protocol, kindly refer to our enclosed publications. On a comparative scale, the above workflow may prove economical.

Best.

Check out mirvana

I use the Mirvana Paris total RNA isolation kit following the miRNA enrichment protocol. We used several kits for isolation and had our genomics dept run each kit on the Bioanalyzer and this is best for miRNAs. For the RT-PCRs I use https://www.quantabio.com/products/microrna-profiling. Lots of helpful info on how to design your assay! In summary (you can read all the info) in cDNA generation, the kit elongates the miRNA with a poly A tail and the reverse primer is universal while the forward primer is basically the miRNA sequence. They have lots of links on QuantaBio including an excel of the forward primers and how to design them. They supply an internal control (SNORD44, similar to GAPDH in mRNA RT-PCRs) but I have better results with RNU6 as my internal control. Im studying sarcomas so my calibrator is U2OS. Im impressed with this method and after optimizing my protocol perfect melt curves and no primer dimers. Good luck!