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.

Life Technologies Ambion® RNA purification products help simplify RNA Extraction for Real-Time PCR. You can also capture your RT-qPCR results directly from cells with Ambion® Cells-to-CT™.

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) isolatedLarge 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 methodFast, convenient silica columnScalable, flexible format with magnetic beadsHighest purity and convenience; includes both organic extraction and silica columnFast, convenient low elution silica column
Preparation time20 minutes<45 minutes30 minutes15 minutes
Amount of starting material10 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)50964050

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.

 

1-20 samples: Qubit® Assays for use on Qubit® Fluorometer

Sku Name Size Price Qty
Q10210 Qubit® RNA BR Assay Kit 100 assays USD 79.00
Q10211 Qubit® RNA BR Assay Kit 500 assays USD 237.00
Q32852 Qubit® RNA HS Assay Kit 100 assays USD 79.00
Q32855 Qubit® RNA HS Assay Kit 500 assays USD 237.00
Q32880 Qubit® microRNA Assay Kit 100 assays USD 83.00
Q32881 Qubit® microRNA Assay Kit 500 assays USD 241.00
Q32866 Qubit® 2.0 Fluorometer 1 each USD 2,035.00

20-2000 samples: Quant-iT™ Assays for use on fluorescent microplate reader

Sku Name Size Price Qty
Q33140 Quant-iT™ RNA Assay Kit 1 kit USD 353.00
Q10213 Quant-iT™ RNA Assay Kit, broad range 1,000 assays USD 339.00
Q32882 Quant-iT™ microRNA Assay Kit 1 kit USD 432.00

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.

Find out more about SuperScript® VILO™ reagent >

 

cDNA Synthesis Kits for Real Time RT-PCR

Sku Name Size Price Qty
11755050 SuperScript® VILO™ Master Mix 50 reactions USD 478.00
11755250 SuperScript® VILO™ Master Mix 250 reactions USD 2,115.00
11755500 SuperScript® VILO™ Master Mix 500 reactions USD 3,525.00
11754050 SuperScript® VILO™ cDNA Synthesis Kit 50 x 20 µL reactions USD 472.00
11754250 SuperScript® VILO™ cDNA Synthesis Kit 250 x 20 µL reactions USD 2,075.00

Reverse Transcriptase

Sku Name Size Price Qty
18080093 SuperScript® III Reverse Transcriptase 2000 units USD 86.50
18080044 SuperScript® III Reverse Transcriptase 10,000 units USD 303.00

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.