Navigation Links
Simple Approaches for Optimization of RT-PCR

Reverse transcription-polymerase chain reaction (RT-PCR) converts and amplifies a single-stranded RNA template to yield abundant double-stranded DNA product(1,2,3). RT-PCR is a powerful tool for analysis of gene expression and for characterization of RNA splice variants. RT-PCR is generally carried out as either one-step RT-PCR, in which all reagents are combined in one tube and RT and PCR are conducted sequentially in this tube(1,3) or two-step RT-PCR, in which RT and PCR are conducted sequentially in separate tubes(2). The USB One-Step RT-PCR Kit (PN 78350), Two-Step RT-PCR Kit (PN 78355), and RT Script Kit (PN 78360) are based on M-MLV Reverse Transcriptase(4) and Taq DNA Polymerase(5). M-MLV Reverse Transcriptase is useful in RT-PCR due to its naturally low RNase H activity(2). The kits are designed for typical routine analyses of gene expression, based on amplification of relatively short (≤ 1.5 kb) RT-PCR products (Fig. 1).

Optimization of reaction parameters is a crucial aspect of RT-PCR. With new targets, optimization can determine the difference between failure and success in generating product. With familiar targets, optimization can minimize use of sample and reagents and maximize reproducibility and reliability of results. A small investment of effort in optimizing reactions can yield a large payoff in results, particularly if careful attention is also paid to fundamental issues such as primer design(6,7), RNA isolation(8,9), and use of RNase free reagents(10). This Tech Tip describes several simple approaches for optimizing sensitivity, specificity, and yield in one-step RT-PCR. The same approaches also apply in two-step RT-PCR.

Method

Reactions (50 μl) were carried out with the USB One-Step RT-PCR Kit (PN 78350) according to the kit protocol, except as no
'"/>

Source:


Page: All 1 2 3 4 5

Related biology technology :

1. Simple, Sensitive, and Rapid Detection of FLAG -Tagged Fusion Proteins
2. A Simple and Rapid Technique to Purify High-Quality Midiprep DNA
3. Simple Purification of DNA from Plasmid Minipreps, PCR Amplifications and Agarose Gels
4. Simple, Sensitive Isotyping of Mouse Monoclonal Antibodies
5. Simple Isolation of RNA from Tissue and Cultured Cells
6. HSVision Molecular Beacon Detection Module Rapidly Detects Herpes Simplex Virus DNA
7. Simple and Rapid Isolation of Plant RNA Using the Eppendorf Perfect RNA, Eukaryotic, Maxi Kit
8. The Fastest, Simplest, and Most Effective Way to Remove DNA Contamination
9. Detection of Morphine and Morphine-Glucuronide in Saliva or Urine with a Rapid and Simple Method Without Derivatization on the Varian 500-MS
10. Gram Quantities of MAb Produced with Simple Bioreactor in Serum-Free Perfusion Culture Replacing Ascitic Fluid Production
11. Two-dimensional Optimization of a Semi-nested PCR for Detecting Listeria monocytogenes

Post Your Comments:
*Name:
*Comment:
*Email:
TAG: Simple Approaches for Optimization PCR

(Date:11/18/2009)...d to be able to find explosive materials and perso... searches, the National Institute of Standards and...nt of Homeland Security, has developed a new certi...al (SRM) 2905, Trace Particulate Explosives. Compa... SRM will be helpful in calibrating, testing and d...
(Date:11/18/2009)..., Nov. 18 Sangamo BioSci...data from the University of Pennsylvania investiga...nc finger nuclease (ZFN) based product, SB-728-T, ...sclosed on the internet. ,, Data were presented...ania School of Medicine from a single subject trea...
(Date:11/18/2009)...est Sussex, U.K. and CLEVELAND, Nov. 18 /PRNewswir...ces business and premier provider of diagnostic sy... labs, has expanded its East Grinstead, U.K., manu... meet increasing global demand for its Sensititre&...cation testing. The expanded facility has been ful...
(Date:11/18/2009)...omega Corporation and SwitchGear Genomics announce...eractions. , Madison, WI,...ion and SwitchGear Genomics announce a novel metho...ults of a study using the HaloCHIP™ System a... ability to correlate DNA binding events to transc...
Breaking Biology Technology:New NIST trace explosives standard slated for homeland security duty 2Sangamo BioSciences Provides Update on Phase 1 Safety Trial of SB-728-T for HIV/AIDS 2TREK Diagnostic Systems Opens New U.K. Facility in East Grinstead to Meet Expanded Customer Demand for Microbiology Products 2TREK Diagnostic Systems Opens New U.K. Facility in East Grinstead to Meet Expanded Customer Demand for Microbiology Products 3TREK Diagnostic Systems Opens New U.K. Facility in East Grinstead to Meet Expanded Customer Demand for Microbiology Products 4Promega and SwitchGear Publish Findings from Combined Technologies that Correlate Binding Events and Transcriptional Activity 2
...o, she was heading to the tiny island nation of T...n the Indian Ocean. By all reports and expectation... coverage. Sixteen days of blissful disconnection....arrived at the tiny Male International Airport , ...d night air. Greeting us on the other side of the ...
...ch of TomoTherapy,s focus has been on sales in I...nce, and the sale is to a hospital that bears the ...on-based medical imaging and device manufacturer h...Hospital , founded in 1909 by Nobel prize winning ... Madame Curie. The Paris institute is comprised of...
...rd. It,s a plane. It,s super hype - able to leap t...nd. Indeed, lately the media has been saturated wi...are sure to result from the mass exodus of Baby Bo...ccounts would lead one to believe that this trend ... But the reality is gray (pun intended). Populatio...
Other Biology Technology:Coveted vacation "disconnect" was elusive 2Coveted vacation "disconnect" was elusive 3Shades of gray: Business impacts of an aging workforce 2Shades of gray: Business impacts of an aging workforce 3Shades of gray: Business impacts of an aging workforce 4Shades of gray: Business impacts of an aging workforce 5Shades of gray: Business impacts of an aging workforce 6
(Date:11/19/2009)...ble farming, initially adopted to preserve soil qu... in maintaining a healthy climate, according to re...and Los Alamos national laboratories. , ORNL and... of the earth,s soils to sequester carbon, with es... greatly reduce U.S. carbon emissions by as much a...
(Date:11/19/2009)...ofessor of paleontology in the McMicken College of...he University of Chicago publish their research in..."Epicontinental Seas Versus Open-Ocean Settings: T..., For many years, paleobiological researchers int...d on charting the many ups (evolutionary radiation...
(Date:11/19/2009)...ofessor in the Fischell Department of Bioengineeri...ence Research and Technology Review Day with a tis...and other types of tissue for implantation. , Fi...nted in an eight minute pitch by faculty members a..., held November 12 at the University of Maryland. ...
Breaking Biology News(10 mins):ORNL, Los Alamos pioneer new approach to assist scientists, farmers 2ORNL, Los Alamos pioneer new approach to assist scientists, farmers 3Paleontologists find extinction rates higher in open-ocean settings during mass extinctions 2Paleontologists find extinction rates higher in open-ocean settings during mass extinctions 3Paleontologists find extinction rates higher in open-ocean settings during mass extinctions 4Bioreactor for bone tissue engineering wins professor venture fair 2Bioreactor for bone tissue engineering wins professor venture fair 3Health Robotics CytoCare 28TM 29 Global Expansion Continues With Grifols International in Latin America 5212 1Health Robotics CytoCare 28TM 29 Global Expansion Continues With Grifols International in Latin America 5212 2Health Robotics CytoCare 28TM 29 Global Expansion Continues With Grifols International in Latin America 5212 3IncentOne Introduces Next Generation of Healthy Choice Award 28TM 29 Incentive Solution to Drive Consumer and Provider Engagement 5209 1IncentOne Introduces Next Generation of Healthy Choice Award 28TM 29 Incentive Solution to Drive Consumer and Provider Engagement 5209 2ESAI provides grant to LA BioMed to investigate long term correction of erectile dysfunction 58855 1ESAI provides grant to LA BioMed to investigate long term correction of erectile dysfunction 58855 2
...lity to eat toxic mulberry leaves without feeling ...understanding why: silkworms contain a special dig...xic chemicals. , Mulberry leaves contain an extre... that break down sucrose (sugar), and thus are pot... called beta-fructofuranosidase is not affected by...
...esearchers from Weill Cornell Medical College may ...of the world,s most common blood disorders, beta-t...ng with sickle-cell anemia, Cooley,s anemia is the...ecting many people of Mediterranean descent, and 2...d Health Organization estimates that between 50,00...
...that a fundamental building block in the cells of ... identically, on a separate branch of the evolutio...ytes that originated at least 420 million years ag...volved from gymnosperms, the group that includes c...lit from lycophytes hundreds of millions of years ...
Other Biology News:Avoiding Spleen Removal for Cooley's Anemia SufferersAvoiding spleen removal for Cooley's anemia suf 2Avoiding Spleen Removal for Cooley's Anemia SufferersAvoiding spleen removal for Cooley's anemia suf 3Avoiding Spleen Removal for Cooley's Anemia SufferersAvoiding spleen removal for Cooley's anemia suf 4When plants 'think' alike 2
Conjugate Stabilizing Diluent HRP from Meridian Life Science, Inc.
...-coil scanning and positioning system is designed ...sitioned or scanned with high velocity and high re...ared to micropositioners with conventional leadscr...n by an integrated, non-contact, zero-friction, vo...
Gram Staining Kit from Sigma-Aldrich
...nthesis and Labeling Kit generates labeled cDNA fo...st unmodified cDNA is made via a reverse transcrip.... Unlike other methods, there are no unnatural mod...n reaction. This unmodified cDNA is then labeled i...
Biology Products: