Real-Time PCR RNA Quantification Workflow
Maximize your RT-qPCR success with optimized starting material
RT-qPCR requires both high-quality RNA as starting material and optimal conversion of the RNA to a DNA copy for accurate real-time PCR detection (qPCR). When an RNA quantitation step is added you’ll be better able to optimize your qPCR analysis.
Learn more about optimizing each step in the workflow by clicking on the tabs below.
The sensitivity and accuracy of RT-qPCR detection depends on the quality and quantity of your input cDNA, which in turn depends on the quality and integrity of the isolated RNA. One important step for avoiding false positives and background is the removal of genomic DNA.
Compare RNA isolation kits for fresh and frozen cells:
|Rapid & relaible method||High-throughput purification||microRNA from most samples||RNA from precious micro-sized samples|
|Order Now||Order Now||Order Now||Order Now|
|PureLink™ RNA Mini Kit||MagMAX™-96 Total RNA Isolation Kit||mirVana™ miRNA Isolation Kit||RNAqueous®-Micro Kit|
|RNA type(s) isolated||Large RNA molecules only (mRNA and rRNA)||Large RNA molecules only (mRNA and rRNA)||Small & large RNA molecules (microRNA, tRNA, mRNA, rRNA)||Small & large RNA molecules (microRNA, tRNA, mRNA, rRNA)|
|Isolation method||Fast, convenient silica column||Scalable, flexible format with magnetic beads||Highest purity and convenience; includes both organic extraction and silica column||Fast, convenient low elution silica column|
|Preparation time||20 minutes||<45 minutes||30 minutes||15 minutes|
|Amount of starting material||10 mg to 100 mg of tissue|
Up to 5 x 107 cells
|Up to 10 mg of tissue|
Up to 100,000 cells
|Up to 100 mg of tissue|
Up to 1 x 107 cells
|Up to 10 mg of tissue
Up to 100,000 cells
|Kit size (preps)||50||96||40||50|
1-20 samples: Qubit® Assays for use on Qubit® Fluorometer
20-2000 samples: Quant-iT™ Assays for use on fluorescent microplate reader
For reliable generation of first strand cDNA, SuperScript® VILO™ cDNA synthesis kit brings you proven SuperScript® III Reverse Transcriptase formulated in an enhanced buffer system. Reduce amplification bias from low- or high-abundance gene products, and capture data that exhibits superior linearity across a broad range of input material.
The SuperScript® VILOTM cDNA kit offers consistent results for your RT experiments and is also available in a convenient single-tube format, facilitating a simplified, time-saving workflow and helping to reduce the risk of contamination.
cDNA Synthesis Kits for Real Time RT-PCR
Deben, C et al. (2013) Expression analysis on archival material revisited: isolation and quantification of RNA extracted from FFPE samples. Diagn Mol Pathol 22(1):59-64.
“A fluorometric analysis is more suitable for quantification of RNA samples extracted from FFPE tissue compared with spectrophotometric analysis." (Qubit® RNA quantitation)
Simbolo, M et al. (2013) DNA qualification workflow for next generation sequencing of histopathological samples. PLoS One 8(6):e62692.
"NanoDrop measured DNA concentrations higher than Qubit and its consistency with dsDNA quantification by qPCR was limited to high molecular weight DNA from FF samples and cell lines, where total DNA and dsDNA quantity virtually coincide. In partially degraded DNA from FFPE samples, only Qubit proved highly reproducible and consistent with qPCR measurements.”