I've seen information where 100-200 ul of plasma was used for miRNA RT-PCR, but if I wanted to do next generation sequencing(NGS) on miRNAs from human plasma, how much plasma do I need to start with to isolate enough RNA for NGS?
Thank you for your assistance.
julie
This is in bovine but they started with 9 ml... DOI: 10.1371/journal.pone.0107259
Please read this paper:http://www.impactaging.com/papers/v5/n10/pdf/100603.pdf
They used 400ul serum to do the miR-Seq.
It depends on the amount of RNA you can recover, typically it is recommended to use between 100ng-1ug of RNA for miRNA sequencing. We have had great results from 100ng of total RNA on our MiSeq system, using the NEB kits for small RNA library prep.
We typically start with 200 µl of serum or plasma and use 100 ng of total RNA as input for small RNA library prep. More information on the miRNA expression workflows in link below.
http://www.biogazelle.com/lab-services/applications/mirna
Very nice paper covering what you asked. We used this paper as a starting point and found it very useful.
Identification of extracellular miRNA in human cerebrospinal
fluid by next-generation sequencing
KASANDRA LOVETTE BURGOS,1,3 ASHKAN JAVAHERIAN,1,3 ROBERTO BOMPREZZI,2 LAYLA GHAFFARI,1
SUSAN RHODES,2 AMANDA COURTRIGHT,1 WAIBHAV TEMBE,1 SEUNGCHAN KIM,1 RAGHU METPALLY,1
and KENDALL VAN KEUREN-JENSEN1,4
1Translational Genomics Research Institute, Phoenix, Arizona 85003, USA
2Barrow Neurological Institute at St. Joseph’s Hospital and Medical Center, Phoenix, Arizona 85013, USA
thank you everyone for responding. We don't have the capability to do the miRNA seq work here, but I am confident I can get 200 ul of plasma from our mice samples!
Greatly appreciate everyone's advice and references,
julie
200 mkl of blood plasma is more than enough. However, currently used methods do not allow efficient cDNA library preparation from plasma miRNA.
Check out the recently published novel NGS library preparation method CATS, which is suited for this application:
https://www.landesbioscience.com/journals/rnabiology/article/29304/?nocache=1646088756
Hi, we obtain >3.5 Million non-duplicate smallRNA reads from only 200 µl with a new sinlge tube method and kit for smallRNA NGS: https://genxpro.net/truequant-smallrna-seq-kit/. This translates into >600 different smallRNA species at >10 reads per million (rpm) and >300 species with >100 rpm (after duplicate removal). This is ~2x more that the best kit tested here: Article Evaluation of commercially available small RNASeq library pr...
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