Real-Time PCR RNA Quantification Workflow | Thermo Fisher Scientific

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.

Invitrogen Ambion RNA purification products from Thermo Fisher Scientific help simplify RNA Extraction for Real-Time PCR. You can also capture your RT-qPCR results directly from cells with Cells-to-C_T.

Compare RNA isolation kits for fresh and frozen cells:

	Rapid & reliable 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

The Qubit™ and Quant-iT™ RNA assays provide specific, sensitive RNA and microRNA quantification, even at low concentrations, with minimal interference from contaminants.

Unlike UV absorbance readings, which are less discriminate, these assays distinguish between DNA, RNA, proteins, and free nucleotides with higher sensitivity than UV absorbance readings, making it possible to measure low-abundance samples. In addition, an intuitive format makes it easier and faster to get your quantifation results.

Find out more about Qubit™ and Quant-iT™ technology for your precious samples.

For reliable generation of first strand cDNA, SuperScript IV VILO Master Mix brings you proven SuperScript IV 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 IV VILO Master Mix is available in a convenient single-tube format, facilitating a simplified, time-saving workflow and helping to reduce the risk of contamination.

Find out more about the SuperScript IV VILO Master Mix ›

RNA Purification

Compare RNA isolation kits for fresh and frozen cells:

	Rapid & reliable 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

RNA Quantification

The Qubit™ and Quant-iT™ RNA assays provide specific, sensitive RNA and microRNA quantification, even at low concentrations, with minimal interference from contaminants.

Find out more about Qubit™ and Quant-iT™ technology for your precious samples.

cDNA Synthesis

The SuperScript IV VILO Master Mix is available in a convenient single-tube format, facilitating a simplified, time-saving workflow and helping to reduce the risk of contamination.

Find out more about the SuperScript IV VILO Master Mix ›

How-to videos

Webinars

Technical resources

Citations

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.”

For Research Use Only. Not for use in diagnostic procedures.