Hi Andrea,

RNA found in both plasma and serum is normally fragmented RNA or small RNA , mainly miRNA, and is either bound to proteins or contained inside extracellular vesicles. If your target is to purify mRNA from plasma/serum then you have to be aware that this mRNA may not be the full length, original RNA, that you may be getting when isolating RNA from whole blood.

I personally recommend for you using one of Norgen's Plasma/Serum RNA Purification kits ( https://norgenbiotek.com/sites/default/files/resources/Plasma-Serum-RNA-Purification-Kit-PI55000-5.pdf). These kits covers a sample volume range from 50ul up to 5mL without using any phenol or carrier RNA. Unlike other purification kits, Norgen's kits uses Silicon carbide as its separating matrix instead of silica. Some literature has mentioned that silica-based technology with/without phenol has a bias towards binding large RNA sizes as well as a bias towards binding RNA with sequences containing high GC contents whereas Norgen's Silicon carbide doesn't have such a bias.

For the isolation of RNA from blood, this should be a straight forward isolation but you have to use a different isolation method for this. I also recommend using Norgen's Total RNA Purificiation kit (Cat. 17200) for this.

When isolating RNA from plasma/serum you should be expecting low RNA amounts (normally in the picogram range) therefore you can't quantify you plasma/serum RNA using conventional methods such as regular spectrometer or Nanodrop since they are not sensitive enough to quantify such low RNA amounts. I recommend using Agilent Bioanalyzer RNA Pico Chip for quantifying plasma/serum RNA. Also don't relay on the RIN values from the bioanalyzer chips cause it will be low and won;t reflect the quality of the purified RNA. RIN values are calculated based on the ratio of the 28S rRNA and the 18S rRNA and since plasma/serum doesn't have cells therefore the purified RNA won't have these two bands which will lead to a very low RIN value. The best way to evaluate the quality of the purified RNA is to amplify a highly abundant small RNA target such as the 5s rRNA or to amplify a highly abundant miRNA such as miR-21. I believe that what Norgen does when performing small RNA sequencing services. https://norgenbiotek.com/services/small-rna-and-microrna-next-gen-sequencing

Sorry for the long message but I just want to give you some detailed information so you don't face any issues through out you project.

I hope this answered all of your concerns.

Best Regards and Good luck.

I use the Qiagen kits. For total RNA, you can use an RNeasy kit. If you want small RNAs, you can use the miRNeasy Serum/Plasma kit.

I use the old exiqon protocol which is a modified version of the miRNeasy kit but they now do their own kits:

http://www.exiqon.com/ls/Documents/Scientific/RNA-Isolation-biofluids-manual.pdf

: http://www.exiqon.com/ls/Documents/Scientific/microRNA-serum-plasma-guidelines.pdf

Plasma/Serum Circulating and Exosomal RNA Purification Mini Kit (Slurry Format) from Norgen Biotek - It can handle samples up to 2mL. (http://norgenbiotek.com/display-product.php?ID=531)

Thanks a lot

We tested the new Exiqon kit and published what we found: it works great, although the mirVana kit is good, too. It helps to use a carrier like highly pure tRNA or phage RNA, linear PA or glycogen.

https://www.researchgate.net/publication/236968834

Article Comparison of Methods for miRNA Extraction from Plasma and Q...

isolation of RNA from human serum and plasma using the Norgen exosomal RNA purification mini kit in order to detect, identify and quantify extracellular RNA.

great

Great suggestions. Thanks

Thanks

Hi all, nice answers, thanks.

I also have a similar question and you might be of help. I collected blood samples from patients and then processed it in order to separate serum, plasma, and whole blood in EDTA tubes.

Do you think RNA extraction kits will be able to extract good quality mRNA? Or did I have to process blood samples differently? Any tip is welcome.

Thanks

Navonil De Sarkar Mohamed Ansar Qureshi Chin Hee Mun Steven Gowelo Kenneth W Witwer Karen Forbes Jennifer I Drake Moemen Abdalla

Hi Andrea,

RNA found in both plasma and serum is normally fragmented RNA or small RNA , mainly miRNA, and is either bound to proteins or contained inside extracellular vesicles. If your target is to purify mRNA from plasma/serum then you have to be aware that this mRNA may not be the full length, original RNA, that you may be getting when isolating RNA from whole blood.

I personally recommend for you using one of Norgen's Plasma/Serum RNA Purification kits ( https://norgenbiotek.com/sites/default/files/resources/Plasma-Serum-RNA-Purification-Kit-PI55000-5.pdf). These kits covers a sample volume range from 50ul up to 5mL without using any phenol or carrier RNA. Unlike other purification kits, Norgen's kits uses Silicon carbide as its separating matrix instead of silica. Some literature has mentioned that silica-based technology with/without phenol has a bias towards binding large RNA sizes as well as a bias towards binding RNA with sequences containing high GC contents whereas Norgen's Silicon carbide doesn't have such a bias.

For the isolation of RNA from blood, this should be a straight forward isolation but you have to use a different isolation method for this. I also recommend using Norgen's Total RNA Purificiation kit (Cat. 17200) for this.

When isolating RNA from plasma/serum you should be expecting low RNA amounts (normally in the picogram range) therefore you can't quantify you plasma/serum RNA using conventional methods such as regular spectrometer or Nanodrop since they are not sensitive enough to quantify such low RNA amounts. I recommend using Agilent Bioanalyzer RNA Pico Chip for quantifying plasma/serum RNA. Also don't relay on the RIN values from the bioanalyzer chips cause it will be low and won;t reflect the quality of the purified RNA. RIN values are calculated based on the ratio of the 28S rRNA and the 18S rRNA and since plasma/serum doesn't have cells therefore the purified RNA won't have these two bands which will lead to a very low RIN value. The best way to evaluate the quality of the purified RNA is to amplify a highly abundant small RNA target such as the 5s rRNA or to amplify a highly abundant miRNA such as miR-21. I believe that what Norgen does when performing small RNA sequencing services. https://norgenbiotek.com/services/small-rna-and-microrna-next-gen-sequencing

Sorry for the long message but I just want to give you some detailed information so you don't face any issues through out you project.

I hope this answered all of your concerns.

Best Regards and Good luck.