Navigation Links
Designing a Successful qRT-PCR Experiment


A Successful One Step qRT-PCR Experiment Requires Careful Consideration of Several Important Factors:

Detection Method: To determine if your gene of interest is represented in an RNA sample, end point PCR followed by gel based staining is suitable. For target quantitation, real-time PCR with SYBR Green I or TaqMan probes are recommended. Many researchers find TaqMan probes to be the most sensitive real-time PCR detection platform. Ambion's MessageSensor RT Kit is compatible with all of these methods.

Designing Primers and Probes: We recommend using primer design software for both TaqMan and SYBR Green I applications to improve your assay design. The software program should accept defining parameters such as melting temperature (Tm) and reaction conditions. Choose the TaqMan probe sequence prior to the primers since probe design is key for successful detection (there are some programs that will choose probe and primer sequences simultaneously such as Primer Express (ABI)). For eukaryotic targets, use primers that overlay an intron-exon junction to eliminate signal from genomic DNA contamination. For best results, amplicon lengths of 80 to 200 bp are recommended.

Primer and Probe Concentrations vs. Target Abundance: Gene-specific primer and TaqMan probe concentrations should be adjusted according to target abundance. By performing a primer titration, an optimal final primer concentration for a given target can be found. This can vary between 150 to 500 nM. An initial concentration of 400 nM for each primer is a useful starting point. The optimal TaqMan probe concentration can also vary according to relative target abundance, though 80 nM is generally a good starting point. We recommend a titration of 50 to 800 nM probe for optimization.

RNA Sample: The quality of the starting RNA template plays an important role in RT-PCR results. Ambion recommends RNAqueous-4PCR to obtain RNA free of DNA. An RNA sample containing contaminating DNA can be treated with Ambion's DNA-free Reagents to help eliminate genomic DNA contamination. To synthesize large amounts of RNA for a standard curve, Ambion recommends the MEGAscript High Yield Transcription Kit.

Control Reactions: Always include at least two controls in your experiments. The first controls for genomic DNA contamination by excluding RT from the reaction (minus RT control). The second, which contains all master mix components except the input RNA, controls for contamination of reagents and the reaction tubes (no-template control). Any other deviations from the master mix will also need appropriate controls.

Master Mix Preparation: A potential source of DNA contamination often arises from prior PCR runs. It is highly recommended that the pre-PCR setup occur at a separate location from post-PCR sample analysis, and that separate pipettes, tips and reaction tubes be used. RT-PCR is highly sensitive to small changes created by pipeting differences and airborne substances. Additionally, Ambion's DNAZap DNA Degradation Solution can be used to decontaminate thermal cyclers, pipettes, and other surfaces prior to RT-PCR.

For a helpful general resource for qRT-PCR approaches and strategies, Ambion recommends the following references: Bustin SA (2000) J Mol Endocrinol 25: 169-93 and Bustin SA (2002) J Mol Endocrinol 29: 23-39.


back to top


Ordering Information
Cat# Product Name Size 1745 MessageSensor RT Kit 50 rxn
'"/>

Source:


Page: All 1 2 3

Related biology technology :

1. Designing a Better siRNA
2. Designing Controls for siRNA Experiments
3. Keys to Successful Densitometry
4. Successful PCR amplification and subcloning of a GC-rich DNA fragment
5. General Considerations for Successful Transfection Experiments
6. Precursor miRNAs for Successful miRNA Functional Studies
7. Recommendations for Successful siRNA Library Screens
8. Setting up Successful siRNA Library Screens
9. Successful stabilization of gene expression profiles
10. HIGH SENSITIVITY qRT-PCR
11. Isolate High Quality Total RNA from LCM Samples Suitable for Microarray and qRT-PCR Analysis
Post Your Comments:
*Name:
*Comment:
*Email:


(Date:10/11/2017)... ... October 11, 2017 , ... The ... endogenous context, enabling overexpression experiments and avoiding the use of exogenous expression plasmids. ... is transformative for performing systematic gain-of-function studies. , This complement to loss-of-function ...
(Date:10/11/2017)... ... October 11, 2017 , ... Proscia ... be hosting a Webinar titled, “Pathology is going digital. Is your lab ready?” ... pathology adoption best practices and how Proscia improves lab economics and realizes an ...
(Date:10/11/2017)... ... October 11, 2017 , ... Disappearing forests and increased emissions are the ... million people each year. Especially those living in larger cities are affected by air ... one of the most pollution-affected countries globally - decided to take action. , “I ...
(Date:10/10/2017)... DIEGO, CALIF. (PRWEB) , ... October 10, 2017 , ... ... as part of its corporate rebranding initiative announced today. The bold new look ... its reach, as the company moves into a significant growth period. , It will ...
Breaking Biology Technology:
(Date:4/17/2017)... Florida , April 17, 2017 NXT-ID, ... technology company, announces the filing of its 2016 Annual Report on ... and Exchange Commission. ... on Form 10-K is available in the Investor Relations section of ... as on the SEC,s website at http://www.sec.gov . ...
(Date:4/11/2017)... Apr. 11, 2017 Research and Markets has ... report to their offering. ... The global eye tracking market to grow at a CAGR of ... Eye Tracking Market 2017-2021, has been prepared based on an in-depth ... market landscape and its growth prospects over the coming years. The ...
(Date:4/5/2017)... SEATTLE , April 5, 2017  The Allen ... the Allen Cell Explorer: a one-of-a-kind portal and dynamic ... large-scale 3D imaging data, the first application of deep ... edited human stem cell lines and a growing suite ... the platform for these and future publicly available resources ...
Breaking Biology News(10 mins):