Navigation Links
RT-PCR procedures

RT-PCR can be performed as either a two-step or an one-step procedure. Each has certain advantages: 1. Two-step procedures A. Two tube, two-step procedure. In the first tube, first-strand cDNA synthesis is performed under optimal conditions, using either random hexamers, oligo(dT) primers (generating a cDNA pool), or sequence-specific primers. An aliquot of the RT reaction is then transferred to another tube (containing thermostable DNA polymerase, DNA polymerase buffer, and PCR primers) for PCR. Advantages of this approach: This method is useful for experiments where multiple transcripts have to be analyzed from the same RT reaction or for specific applications such as Differential Display Reverse Transcription (DDRT) or Rapid Amplification of cDNA Ends (RACE). Also, since the RT reaction is performed under optimal conditions, this approach produces the longest RT-PCR products (up to 14 kb in length, if the appropriate enzymes are used). B. One tube, two-step procedure. In the first step, reverse transcriptase produces first-strand cDNA in the presence of Mg2+ ions, high concentrations of dNTPs, and either specific or non-specific [oligo(dT)] primers (reaction volume, 20 l). Following the RT reaction, an optimized PCR buffer (without Mg2+ ions), a thermostable DNA polymerase, and specific primers are added to the tube and PCR is performed. This approach may be useful when template amounts are limited, since the entire RT reaction is used in the subsequent PCR. Advantages of Two-step RT-PCR Procedure. A two-step procedure has the following advantages:
  • Optimizes reaction conditions. The two-step format allows both reverse transcription and PCR to be performed under optimal conditions to ensure efficient and accurate amplification.
  • Provides flexibility. Two-step procedures allow the product of a single cDNA synthesis reaction to be used for analysis of multiple transcripts. This flexibility is valuable for such specialized applications as rapid amplification of cDNA ends (RACE) and Differential Display Reverse Transcription (DDRT).
  • Amplifies long sequences. With the right combination of reverse transcriptase and thermostable DNA polymerase, two-step RT-PCR can amplify RNA sequences up to 14 kb long.
2. One tube, one-step procedure (coupled RT-PCR) Both cDNA synthesis and PCR amplification are performed with the same buffer and site-specific primers, eliminating the need to open the reaction tube between the RT and PCR steps. In addition to the higher sensitivity of this approach (as in the one tube, two-step reaction above), the one-step approach minimizes the chance of contamination, since the entire reaction is performed with minimal pipetting steps and without opening the tube. In addition, this approach permits direct analysis of a specific transcript, since the primers used in both steps are sequence-specific. Finally, the thermoactive reverse transcriptase used in this procedure allow a high RT reaction temperature (5072C), which reduces false priming and increases the specificity of the reaction by eliminating secondary mRNA structure. Advantages of One-step RT-PCR Procedure. A one-step procedure has the following advantages:
  • Minimizes time required. The one-step reaction has fewer pipetting steps than the two-step reaction, significantly reducing the time needed to perform RT-PCR and eliminating pipetting errors.
  • Reduces the risk of contamination. The entire one-step reaction takes place in a single tube, with no transfers required and no need to open the reaction tube (steps where contamination of the PCR sample can take place).
  • Improves the sensitivity and specificity of cDNA synthesis. Two characteristics of the one-step reaction provide increased yield and efficiency: (1) the cDNA reaction is performed at a high temperature (to eliminate problems with RNA secondary structure), and (2) the entire cDNA sample is used as template for PCR.



Page: All 1 2 3

Related biology technology :

1. New Kit Generates Exceptionally Pure Total RNA for RT-PCR
2. Brilliant Core Reagent Kits for QPCR and QRT-PCR
3. Control RT-PCR Primers for Human Gene Transcripts with Varying Abundance
4. prostar RT-PCR Systems for Robust High-Fidelity RNA Amplification
5. Performance Comparisons of Commercial RT-PCR Systems
6. RT-PCR Primer Sets for Human and Mouse Mismatch Repair Genes
7. RT-PCR Primers for the Study of Apoptosis
8. cMaster RTplusPCR System for increased sensitivity and product length in RT-PCR
9. Single-cell RT-PCR Analysis of Paramecium Primaurelia with the Eppendorf cMaster RTplusPCR System
10. Optimized Dengue RT-PCR
11. QuantiTect SYBR Green RT-PCR Kit
Post Your Comments:

(Date:12/1/2015)... Texas (PRWEB) , ... December ... ... , a leading relationship marketing company specializing in scientifically backed, age-defying products, ... January 2016 issue, which highlights the exponential success and unrivaled opportunities that ...
(Date:12/1/2015)... (PRWEB) , ... December 01, 2015 , ... ... global meeting this month and Dr. J. Kyle Mathews will join ... includes the new single site hysterectomy. , An experienced urogynecologist, founder of Plano ...
(Date:11/30/2015)... ... ... Global Stem Cells Group announced that its scientific team is in the ... cells. The announcement starts a new phase toward launching the simple, quick system for ... lipoaspirate obtained from liposuction of excess adipose tissue. , Lipoaspirate, contains a large ...
(Date:11/30/2015)... MONTREAL , Dec. 1, 2015 /PRNewswire/ - BioAmber Inc. (NYSE: ... that it has joined the American Business Act on Climate ... economy that are standing with the Obama Administration to demonstrate ... for a strong outcome to the COP21 Paris ... . --> Sarnia, Canada . ...
Breaking Biology Technology:
(Date:11/17/2015)... Nov. 17, 2015  Vigilant Solutions announces today that ... Board of Directors. --> ... retiring from the partnership at TPG Capital, one of ... over $140 Billion in revenue.  He founded and led ... the TPG companies, from 1997 to 2013.  In his ...
(Date:11/16/2015)... , Nov 16, 2015  Synaptics Inc. ... human interface solutions, today announced expansion of its ... ™ touch controller and display driver integration ... of smartphones. These new TDDI products add to ... (HD resolution), TD4302 (WQHD resolution), and TD4322 (FHD ...
(Date:11/12/2015)... 2015  Arxspan has entered into an agreement ... for use of its ArxLab cloud-based suite of ... partnership will support the institute,s efforts to electronically ... information internally and with external collaborators. The ArxLab ... the Institute,s electronic laboratory notebook, compound and assay ...
Breaking Biology News(10 mins):