Background

Efficient extraction of high-quality (concentration: 800–2,200 ng/μL) and high molecular weight (>50 kb) genomic DNA and tissue RNA from the limited amount of available samples is the key challenge for downstream applications like next-generation DNA sequencing (NGS) or transcriptomics studies, among others. The expanding research on human diseases related to gut microbiota and associated gut health resulted in a plethora of methodologies used to profile the gut microbial composition and associated inflammatory status of the gut tissue.

Each step in the downstream data analysis process is subject to technical variation depending on the protocol or materials used for DNA or RNA isolation. Researchers have found striking differences in the results obtained from different kit-based methods within the same laboratory (Ferrand et al., 2014; Videnska et al., 2019; Gryp et al., 2020). Therefore, standardizing the protocols is of utmost priority for researchers to obtain consistent, good quality, and robust representative data.

The diversity of the gut microbes in gut ecosystems can be categorized based on their morphological, structural, biochemical, and genetic properties. Gut microbes also have notable differences regarding cell wall structure and cell membranes, which enclose their cytoplasm and genomic contents. Harsh sample treatments can affect DNA quality, while a mild process may cause partial lysis of the microbial community (Bag et al., 2016). Moreover, the unknown composition of the lysis buffer of kit-based methods presents different biases, drastically changing the results obtained from the same sample (Brooks et al., 2015). Therefore, it is important to optimize cell lysis methods to obtain genomic DNA from all groups of microbes present in the gut.

We developed a very efficient, cost-effective cell lysis method. Reagents used to standardize the process are readily available, even in a comparatively less equipped laboratory, and the reagents are not too expensive. The yield and quality of the DNA are also better than those obtained by the readily available kit-based methods.

In an RNA isolation procedure from gut tissue, it is essential to standardize the amount of tissue required to obtain good quality RNA. Proper tissue homogenization is another crucial step for a high yield of RNA. To get high quality and quantity of RNA, we standardized the needed amount of colonic tissue and the liquid nitrogen-based homogenization process.

These methodologies were published in a recent article (Pradhan et al., 2022).