Effective RNA Extraction for Real-Time RT-PCR: Methods and Solutions

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March 30, 2020 Science

RNA extraction for detection and analysis of viral RNA with RT-PCR - a comparison of different extraction methods

The extraction and isolation of viral RNA is the first and most crucial step in detecting viral RNA via real-time RT-PCR. In this article, we present proven methods for successfully extracting viral RNA.

According to the Robert Koch Institute, nested RT-PCR and real-time RT-PCR are established methods for the analysis of viral infections. Unlike ELISA or IFA, these methods can be performed earlier during the infection. For real-time RT-PCR to provide accurate results, viral RNA must be efficiently isolated first.

The most common RNA extraction methods include:

Purification kits and spin columns
Single-step method by Chomczynski and Sacchi
Classic phenol-chloroform method

Although all these methods are generally efficient, a common issue is the remaining genomic DNA after RNA extraction. This DNA contamination can lead to false-positive results, especially in subsequent applications such as RT-PCR. To avoid this, genomic DNA should be removed via DNase I treatment or phenol-chloroform extraction. Additionally, using an RNase inhibitor can prevent RNA degradation during the process.

1. Extraction of Viral RNA with Commercial Purification Kits

The extraction of viral RNA using commercial purification kits is based on the principle of “spin column-based nucleic acid purification.” This approach uses a lysis buffer to prepare the cells and apply the homogenate to special purification columns. The chaotropic salts in the lysis buffer inactivate any present RNases.

The Total RNA Purification Kit – Tissue from Genaxxon allows the extraction of high-quality RNA from tissues (also available for blood/cell cultures). This kit is specifically designed for RNA isolation, utilizing mini spin columns with membranes that efficiently and selectively bind nucleic acids in the presence of high concentrations of chaotropic salts. RNases are inactivated by guanidinium thiocyanate and β-mercaptoethanol.

Ceramic beads, available separately, can further improve homogenization. Additionally, Genaxxon offers RNA Mini Spin Purification Columns compatible with other RNA purification kits from various suppliers.

2. RNA Extraction Using the Single-Step Method (Chomczynski and Sacchi) with GENAzol

The single-step method by Chomczynski and Sacchi is an adaptation of the two-phase method, utilizing GENAzol, a reagent containing guanidinium thiocyanate and phenol. Guanidinium thiocyanate acts as a denaturant, lysing cells and inhibiting RNases, keeping RNA stable. In the organic phenol phase, proteins and DNA dissolve, while RNA remains in the aqueous phase.

After homogenizing the sample with GENAzol, chloroform is added, causing phase separation into three layers: a clear aqueous upper phase containing RNA, an interphase, and an organic lower phase containing DNA and proteins. RNA is precipitated from the aqueous phase by adding isopropanol, washed to remove impurities, and then resuspended for further applications.

3. Homemade Cell Lysis and Extraction Buffer Solutions

Due to supply shortages of commercially available RNA isolation kits and their components, homemade buffer solutions can be a viable alternative for laboratories. Studies have shown that these homemade solutions, in combination with traditional purification kits, deliver comparable results for qRT-PCR. A study by the Friedrich-Schiller-University Jena (Emmer et al. 2020) evaluated simple-to-prepare buffer solutions for nucleic acid extraction from respiratory material of SARS-CoV-2-positive patients.

a. Buffer solutions based on denaturing and chaotropic substances like guanidinium thiocyanate:

Buffer solution with 2-mercaptoethanol: This solution contains 2-mercaptoethanol, which requires special safety measures due to its toxicity, including protective clothing and working under a fume hood.
Buffer solution without 2-mercaptoethanol: This alternative lacks toxic 2-mercaptoethanol but shows a slightly higher Cq value. The octylphenol derivative Triton X-100 it contains is classified as a substance of very high concern due to its endocrine-disrupting effects.

b. Guanidinium thiocyanate-free buffer solutions:

These buffers are less efficient compared to those with guanidinium thiocyanate. When the potent guanidinium salt is unavailable, the following alternatives can be used:

Buffer solution with Triton X: Triton X-100 is classified as a substance of very high concern due to its endocrine-disrupting effects.
SDS lysis buffer: A protocol for the following real-time RT-PCR was provided by the Charité in Berlin, recognized by the WHO as a reference laboratory for the investigation of SARS-CoV-2 viruses. You can find the protocol here: WHO Protocol.

From RNA Isolation to Amplification: Efficient Solutions for RT-PCR

After successfully extracting RNA, the next step is the reverse transcription and amplification of viral RNA through RT-PCR, a crucial process for accurate detection and quantification of the target RNA. To optimize this process, Genaxxon offers two specialized products that enhance both efficiency and accuracy.

The HotScriptase RT Mastermix from Genaxxon offers a unique solution for simultaneous reverse transcription and DNA amplification in one step. By combining reverse transcriptase and DNA polymerase in a single enzyme, the usual isothermal intermediate step is eliminated, saving both time and costs. This innovative approach reduces pipetting steps, minimizing contamination risks and ensuring reliable results.

For more demanding applications, such as synthesizing longer cDNAs (up to 17 kb) or working with GC-rich target RNAs, the Scriptase RT-cDNA synthesis kit from Genaxxon is an excellent choice. This proprietary reverse transcriptase is characterized by reduced RNase H activity and increased thermostability, making it ideal for applications like RNA-seq, gene expression analysis, and diagnostics. With Oligo(dT) and random hexamer primers included, the Scriptase RT-cDNA synthesis kit offers flexibility and guarantees high sensitivity in cDNA synthesis.