One-Step RT-PCR Kit Vs. Two-Step RT-PCR Kit: Detailed Differences

Often individuals get confused between choosing One-Step RT-PCR Kit and Two-Step RT-PCR kit. Well, both come with their unique set of advantages and uses. In this blog, you will get to learn about both setups in a detailed description.

Comparison between One-Step RT-PCR Kit and Two-Step RT-PCR Kit

Here's a concrete comparison between both setups, allowing you to decide which one's more suitable for your requirements:

1.      One-Step RT-PCR Kit

The One-Step RT-PCR Kit consists of a single reaction tube, the first-strand cDNA synthesis (RT) and subsequent PCR. This kit setup makes the workflow much easier by greatly reducing the possibilities of variation. It also works to minimize the chances of contamination. This kit setup allows high-throughput applications and hence can be used for a large number of samples. For the amplification procedure, this kit involves using gene-specific primers. As a result, the results for gene analysis per RNA sample are limited. You will also require efficient protein ladders for further quantification of the DNA fragments.

Setup

It comes with the setup of combined reactions under conditions facilitating both reverse transcription and PCR.

Primers

It makes the use of Gene-specific primers. These are components that can be readily utilized to detect specific genes. With Gene-specific primers, you are no longer required to access manual primer design.

Use

The ideal use of this kit setup is to analyze specific genes through high-throughput platforms.

Advantages

The advantages of using this kit setup are as follows:

●       Less experimental variation

As both reactions take place in the same tube, there's less scope for experimental variation, thereby increasing the accuracy of the results.

●       Reduced risks of contamination

This kit setup involves less number of pipetting steps. Therefore, the risks of contamination are greatly minimized.

●       Facilitates high throughput amplification

This type of setup is highly suitable for high throughput amplification or screening, making it suitable to use for large samples.

2.      Two-Step RT-PCR Kit

The Two-Step RT-PCR Kit comprises first-strand cDNA synthesis (RT) that is later followed by an amplification procedure of a portion of the resulting cDNA by PCR in a distinct tube. Therefore, from a single RNA sample, this kit can detect multiple genes. Moreover, since the RT and PCR reactions are separated in this case, it provides a better scope for optimizing reaction conditions and enhances flexibility. The presence of an efficient RNase Inhibitor can further facilitate the procedure of gene detection.

Setup

It comes with a separate setup for optimized reactions of both reverse transcription and PCR.

Primers

Random hexamers, oligo (dT), are usually used. Gene-specific primers can also be utilized.

Use

The ideal use of this kit setup is the analysis of multiple genes. TNF-alpha can be used to propagate the reaction appropriately.

Advantages

The advantages of this kit setup are stated below:

●       Supports multiple reactions for a long time

Since the stable cDNA pool can be preserved for a long time, it can facilitate multiple reactions in the long run.

●       No requirement for multiplexing

Since the target and reference genes can undergo amplification from the same pool, there will be no need for multiplexing.

●       Flexible pricing options

This setup offers ideal and flexible priming options.

Final words

This was a comprehensive comparison between both the kit setups. Now, depending upon your requirements, you can cater your purchase. If you want to get the best biotech products such as Taq DNA Polymerase, protein markers, etc., you can consult Universal Biotechnology.