RNA quantification Fuorimetric

RNA quantification for appropriate concentration and quality (260/280 ratio) is an important step before downstream analysis (including sequencing, RT-qPCR, etc.). Having insufficient RNA quantities or a high salt or phenol in the RNA product can lead to variable or irreproducible downstream results. The various methods used for RNA quantification include: 1. UV spectrophotometric (challenges include: low sensitivity, cannot distinguish between nucleic acid species), 2. Fluorescence-based (challenges include: requires standards, cannot measure amplifiability, not sequence-specific), and 3. RT-PCR (challenges include: requires standards, time-intensive, costly). In order to overcome these challenges, and also to ensure the proper quantification and quality control for RNA product, it is important to use at least two or more methods in order to discard any inconsistencies. Using standards for calibrations increases the sensitivity range for RNA detention (fluorescence- and RT-PCR-based methods). When using RT- PCR, it is important to choose correct primers, aligning to the desired site on the template and of appropriate product length, along with positive, negative and loading controls. It is also important to have at least two primer pairs in order to confirm results.

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Found 5 matching solutions for this experiment

Protocol tips
The amount of total RNA was determined using QuantiFluor RNA system (Promega, WI, USA). For detection of cellular RNA, miRNeasy kit (217004, Qiagen, Venlo, Netherlands) was used in the extraction step.
Downstream tips
MiRNA was then analysed with the same method as described above, and the data was normalised to the expression of U6 snRNA. For analysis of mRNA or primary miRNA, extracted RNA was subjected to cDNA synthesis using PrimeScript RT Master Mix (TAKARA, Shiga, Japan) and PCR was performed using TaqMan Fast Advanced Master Mix and StepOnePlus realtime PCR system. The data was normalised to the expression of GAPDH (glyceraldehyde-3-phosphate dehydrogenase).
Qubit® RNA HS Assay Kit

Thermo Fisher Scientific

Protocol tips
All RNA samples were treated with DNase I in solution to completely remove genomic DNA. RNA samples were quantified with a Qubit 2.0 (RNA HS kit; Thermo) and were then analyzed on the BioAnalyzer High Sensitivity DNA chip (Agilent) to determine RNA integrity before library construction.
Agilent RNA 6000 Pico Kit

Agilent Technologies

Protocol tips
RNA 6000 Pico Chip, Bioanalyzer: detection of intact ribosomal RNA peak will make the exclusion of the sample for further analysis, since will evidence of residual cell contamination (eukaryotic: 18S (1869 nt), 28S rRNA (5070 nt); prokaryotic: 16S (1542 nt), 23S rRNA (2906 nt)).
Protocol tips
otal RNA concentration and total cDNA concentration were quantified by the RiboGreen RNA Quantitation Kit and PicoGreen dsDNA Quantitation Kit, respectively (Chang et al., 2006).
Protocol tips
Expression levels of target RNA were normalized to total RNA quantified using Quant-iT RiboGreen RNA Reagent (Thermo Fisher Scientific).
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