title: Lowering the quantification limit of the QubitTM RNA HS assay using RNA spike-in.
abstract: BACKGROUND
  RNA quantification is often a prerequisite for most RNA analyses such as RNA sequencing. However, the relatively low sensitivity and large sample consumption of traditional RNA quantification methods such as UV spectrophotometry and even the much more sensitive fluorescence-based RNA quantification assays, such as the Qubit™ RNA HS Assay, are often inadequate for measuring minute levels of RNA isolated from limited cell and tissue samples and biofluids. Thus, there is a pressing need for a more sensitive method to reliably and robustly detect trace levels of RNA without interference from DNA.
  
  
  METHODS
  To improve the quantification limit of the Qubit™ RNA HS Assay, we spiked-in a known quantity of RNA to achieve the minimum reading required by the assay. Samples containing trace amounts of RNA were then added to the spike-in and measured as a reading increase over RNA spike-in baseline. We determined the accuracy and precision of reading increases between 1 and 20 pg/μL as well as RNA-specificity in this range, and compared to those of RiboGreen(®), another sensitive fluorescence-based RNA quantification assay. We then applied Qubit™ Assay with RNA spike-in to quantify plasma RNA samples.
  
  
  RESULTS
  RNA spike-in improved the quantification limit of the Qubit™ RNA HS Assay 5-fold, from 25 pg/μL down to 5 pg/μL while maintaining high specificity to RNA. This enabled quantification of RNA with original concentration as low as 55.6 pg/μL compared to 250 pg/μL for the standard assay and decreased sample consumption from 5 to 1 ng. Plasma RNA samples that were not measurable by the Qubit™ RNA HS Assay were measurable by our modified method.
  
  
  CONCLUSIONS
  The Qubit™ RNA HS Assay with RNA spike-in is able to quantify RNA with high specificity at 5-fold lower concentration and uses 5-fold less sample quantity than the standard Qubit™ Assay.
authors.affiliation: Department of Obstetrics & Gynecology and Women's Health, Albert Einstein College of Medicine, 10461, Bronx, NY, USA. xin.li@einstein.yu.edu.|Department of Genetics, Albert Einstein College of Medicine, 10461, Bronx, NY, USA. xin.li@einstein.yu.edu.
authors.forename: Xin
authors.initials: X
authors.lastname: Li
authors.affiliation: Nephrology and Hypertension, Hadassah - Hebrew University Medical Center, 91120, Jerusalem, Israel. iddo@hadassah.org.il.
authors.forename: Iddo Z
authors.initials: IZ
authors.lastname: Ben-Dov
authors.affiliation: Department of Obstetrics & Gynecology and Women's Health, Albert Einstein College of Medicine, 10461, Bronx, NY, USA. maurizio.mauro@einstein.yu.edu.|Department of Genetics, Albert Einstein College of Medicine, 10461, Bronx, NY, USA. maurizio.mauro@einstein.yu.edu.
authors.forename: Maurizio
authors.initials: M
authors.lastname: Mauro
authors.affiliation: Department of Obstetrics & Gynecology and Women's Health, Albert Einstein College of Medicine, 10461, Bronx, NY, USA. zev.williams@einstein.yu.edu.|Department of Genetics, Albert Einstein College of Medicine, 10461, Bronx, NY, USA. zev.williams@einstein.yu.edu.
authors.forename: Zev
authors.initials: Z
authors.lastname: Williams
mesh_terms: D002623:Chemistry Techniques, Analytical / Q000379:methods*; D006801:Humans; D012313:RNA / Q000032:analysis* / Q000097:blood; D015203:Reproducibility of Results; D013045:Species Specificity
chemical_list: D012313:RNA