Fast and accurate qPCR with SensiFast

Fast and accurate qPCR with SensiFast, qpcr | Medical Supply Company

 One Step versus Two Step real time PCR, which should you use? 

The short answer is, there is no short answer, both systems have their advantages and disadvantages. 

First of all let’s take a look at how the two techniques work: 

One Step real time PCR starts with addition of RNA, gene-specific primers and a one step real-time PCR mix added to one tube. The first reaction which takes place in this tube is reverse transcription of the RNA which the gene-specific primers have bound to, this is immediately followed by real-time PCR cycles to produce PCR products.  

Two Step real time PCR requires two separate reactions. In the first step, RNA, primers (Oligo dT or random hexamers) and a cDNA synthesis mix containing reverse transcriptase and dNTPs are mixed and a specific thermal cycle used to reverse transcribe the RNA to first strand cDNA. This cDNA is then added to a second reaction mix containing gene specific primers and real-time PCR mix. The real-time PCR is carried out on a specific cycler such as the BMS Mic.   

Pros and Cons of each method 

One Step qPCR 

Pros Cons 
Accurate representation of target copy number Often less sensitive 
Simple and rapid Difficult to troubleshoot RT step 
Fewer pipetting steps No stock of cDNA produced  
Best option for high throughput screening  
Best option when a small number of assays will be run repeatedly  
Multiplex gene of interest and control in same well  

Two Step qPCR 

Pros Cons 
User can independently optimise both reactions to increase efficiency Time consuming 
Highly sensitive More pipetting steps 
More efficient More optimisation required 
Stock cDNA produced to allow quantification of multiple targets  
Ideal when starting material is limited  

Which method should I use for my experiment? 

One Step qPCR is useful when analysing a few genes over a large number of samples, Two Step qPCR is useful when analysing a large number of genes over fewer samples. 

In general, Two Step qPCR is more sensitive as the reverse transcription and PCR reactions can be optimised individually, also the cDNA produced is more stable than RNA allowing for easier archiving. While One Step qPCR is ideally suited for high throughput screening experiments where less pipetting steps to reduce variation and potential contamination are very important. 

Bioline One-Step and Two-Step qPCR reagents available from MSC 

MSC supply the Bioline SensiFAST range of reagents for both One-Step and Two-Step qPCR.

One step qPCR ReagentsTwo Step qPCR Reagents
Fast qPCR reagents for both SYBR and Probe qPCR chemistry  cDNA synthesis kit with mixed OligodT and random primers for enhanced coverage  
Optimised buffers for superior sensitivity and specificity  Fast qPCR reagents for both SYBR and Probe qPCR chemistry  
Results in as little as 40 minutesExcellent for low abundance targets
Minimised primer-dimer effects   

  

Contact your local MSC sales rep for more information. 

Leave a Reply

Your email address will not be published. Required fields are marked *