RNA quantification qPCR

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 3 matching solutions for this experiment

Protocol tips
The main characteristics of the different assays were as follows: (1) the laboratory associated with the NRC in-house consensus HDV real-time RT-PCR method (NRC-hepatitis delta virus quantification [HDVQ]), (2) the Roche Lightmix HDV kit (Lightmix Roche), (3) the Robogene HDV RNA quantification kit (Aj-Roboscreen), and (4) the DiaPro HDV RNA Quantification (QT) (DiaPro) are summarized in Table 1.
Protocol tips
Prepare standard 20 uL final reaction mix.

Reverse transcribe at 45°C for 10 min, initiate polymerase activation at 95°C for 2min
Downstream tips
If PCR efficiency below 90% Increase extension time/Increase concentration of primers in 100nM increments
Upstream tips
After reverse transcription,start the PCR with an initial incubation step of 5 min at 95°C to activate HotStarTaq Plus DNA Polymerase.
Protocol tips
Program the real-time cycler according to the product protocol
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