Molecular Biology

Circular RNA (circRNA) is an intriguing class of non-coding RNA that exists as a continuous closed loop. With the improvements in high throughput sequencing, biochemical analysis, and bioinformatic algorithms, studies on circRNA expression became abundant in recent years. However, functional studies of circRNA are still limited. Subcellular localization of circRNA may provide some clues in elucidating its biological functions by performing subcellular fractionation assay. Notably, circRNAs that are predominantly found in the cytoplasm are more likely to be involved in post-transcriptional gene regulation, e.g., acting as micoRNA sponge, whereas nuclear-retained circRNAs are predicted to play a role in transcriptional regulation. Subcellular fractionation could help researchers to narrow down and prioritize downstream experiments. The majority of the currently available protocols describe the steps for subcellular fractionation followed by western blot analysis for protein molecules. Here, we present a protocol for the subcellular fractionation of cells to detect circRNA via RT-qPCR with divergent primers. Moreover, detailed steps for the generation of specific circRNAs-enriched cDNA included in this protocol will enhance the amplification and detection of low-abundance circRNAs. This will be useful for researchers studying low-abundance circRNAs.

Key features

• This protocol builds upon the method developed by Gagnon et al. (2014) and extends its application to circRNA study.

• Protocol for amplification of low levels of circRNA expression.

• Analysis takes into consideration the ratio of cytoplasmic RNA concentration to nuclear RNA concentration.

Graphical overview

When performing expression analysis either for coding RNA (e.g., mRNA) or non-coding RNA (e.g., miRNA), reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) is a widely used method. To normalize these data, one or more stable endogenous references must be identified. RefFinder is an online web-based tool using four almost universally used algorithms for assessing candidate endogenous references—delta-Ct, BestKeeper, geNorm, and Normfinder. However, the online interface is presently cumbersome and time consuming. We developed an R package, RefSeeker, which performs easy and straightforward RefFinder analysis by enabling raw data import and calculation of stability from each of the algorithms and provides data output tools to create graphs and tables. This protocol uses RefSeeker R package for fast and simple RefFinder stability analysis.

Key features

• Perform stability analysis using five algorithms: Normfinder, geNorm, delta-Ct, BestKeeper, and RefFinder.

• Identification of endogenous references for normalization of RT-qPCR data.

• Create publication-ready graphs and tables output.

• Step-by-step guide dialog window for novice R users.

Graphical overview

Simple workflow diagram. Two main workflow paths are presented. A) Using the RefSeeker wizard allows non-R programmers to easily load data and choose between selected output formats. B) Command line interface provides more options to control input and output formats and to automate analysis.

The nematode Haemonchus placei is a pathogenic parasite, the most seriously affecting ruminant’s health and being responsible for enormous economic losses all over the world. The present protocol describes different in vitro techniques to select potential candidate antigens with immune-protective activity from excretory and secretory products (ESP) from H. placei transitory infective larvae (xL₃). ESP from xL₃ were obtained from the in vitro infective larvae (L₃) maintained in Hank’s medium at 37 °C with 5% CO₂ for 48 h. Then, the presence of ESP proteins was confirmed by SDS-PAGE to be used in an in vitro proliferation assay with bovine peripheral blood mononuclear cells (PBMCs). The ESP were exposed to the PBMCs during two different periods (24 and 48 h). Genes associated with immune response against the nematode were analyzed using relative gene expression and bioinformatic tools. These are simple, economic, and helpful tools to identify potential immune-protective molecules under in vitro conditions for confirming the efficacy of future in vivo assays.

Graphical overview

E-cigarette (E-cig) inhalation affects health status by modulating inflammation profiles in several organs, including the brain, lung, heart, and colon. The effect of flavored fourth-generation pod-based E-cigs (JUUL) on murine gut inflammation is modulated by both flavor and exposure period. Exposure of mice to JUUL mango and JUUL mint for one month upregulated inflammatory cytokines, particularly TNF-α, IL-6, and Cxcl-1 (IL-8). JUUL Mango effects were more prominent than those incurred by JUUL Mint after one month of exposure. However, JUUL Mango reduced the expression of colonic inflammatory cytokines after three months of exposure. In this protocol, we detail the process of RNA isolation from the mouse colon and the use of extracted RNA in profiling the inflammatory milieu. Efficient RNA extraction from the murine colon is the most important step in the evaluation of inflammatory transcripts in the colon.

The quantification of plant hormones and related gene expression is essential to improve the understanding of the molecular regulation of plant growth and development. However, plant hormone quantification is still challenging due to extremely low endogenous levels and high chemical diversity. In this study, we present a convenient extraction protocol that enables the simultaneous extraction of both phytohormones and RNA from the same sample in a small quantity (approximately 10 mg). Using ultra-performance liquid chromatography coupled with tandem mass spectrometry (UPLC–MS/MS), this protocol provides a method to quantify 13 phytohormones and their derivatives from four classes (cytokinin, auxin, abscisic acid, and gibberellin) at the speed of 14 min per sample.

The impact of viral diseases on human health is becoming increasingly prevalent globally with the burden of disease being shared between resource-rich and poor areas. As seen in the global pandemic caused by SARS-CoV-2, there is a need to establish viral detection techniques applicable to resource-limited areas that provide sensitive and specific testing with a logistically conscious mindset. Herein, we describe a direct-to-PCR technology utilizing mechanical homogenization prior to viral PCR detection, which allows the user to bypass traditional RNA extraction techniques for accurate detection of human coronavirus. This methodology was validated in vitro, utilizing human coronavirus 229E (HCoV-229E), and then clinically, utilizing patient samples to test for SARS-CoV-2 infection. In this manuscript, we describe in detail the protocol utilized to determine the limit of detection for this methodology with in vitro testing of HCoV-229E.

Cyclic diguanylate monophosphate (c-di-GMP) is a second messenger signaling molecule that drives the transition from planktonic to the biofilm mode of growth in many bacterial species. Pseudomonas aeruginosa has at least two surface sensing systems that produce c-di-GMP in response to surface attachment, the Wsp and Pil-Chp systems. We recently used a plasmid-based c-di-GMP reporter (pP_cdrA::gfp) to describe how the Wsp system generates heterogeneity in surface sensing, resulting in two physiologically distinct subpopulations of cells during early biofilm formation. One subpopulation has elevated c-di-GMP and produces biofilm matrix, serving as the founders of initial microcolonies. The other subpopulation has low c-di-GMP and engages in surface motility, allowing for exploration of the surface. Here, we describe the protocol for a key experiment to confirm our initial observation of c-di-GMP heterogeneity during surface sensing: the use of flow-assisted cell sorting (FACS) to isolate subpopulations of cells with high and low c-di-GMP reporter activity, followed by quantitative Reverse Transcriptase PCR (qRT-PCR) of genes that are known to be transcriptionally regulated in response to cellular c-di-GMP levels (pelA, pslA). This protocol can be adapted by others to isolate subpopulations of high- and low- c-di-GMP P. aeruginosa cells that are genetically identical, but phenotypically distinct for future experiments examining specific mRNA transcripts as we did or, presumably, for additional applications like RNAseq, proteomics, or TNseq.

Graphical abstract

Given the scale of the ongoing COVID-19 pandemic, the need for reliable, scalable testing, and the likelihood of reagent shortages, especially in resource-poor settings, we have developed an RT-qPCR assay that relies on an alternative to conventional viral reverse transcriptases, a thermostable reverse transcriptase/DNA polymerase (RTX) (Ellefson et al., 2016). Here we show that RTX performs comparably to the other assays sanctioned by the CDC and validated in kit format. We demonstrate two modes of RTX use – (i) dye-based RT-qPCR assays that require only RTX polymerase, and (ii) TaqMan RT-qPCR assays that use a combination of RTX and Taq DNA polymerases (as the RTX exonuclease does not degrade a TaqMan probe). We also provide straightforward recipes for the purification of this alternative reagent RTX. We anticipate that in low resource or point-of-need settings researchers could obtain the available constructs and begin to develop their own assays, within whatever regulatory framework exists for them.

The single-cell transcriptome is the set of messenger RNA molecules expressed in one cell. It is extremely variable and changes according to external, physical and biochemical conditions. Due to sensitivity shortages, most of genetic studies use bulk samples, providing only the average gene expression. Single-cell technologies have provided a powerful approach to a more detailed understanding of the heterogenic populations and minority cells. However, since it is still a quite novel technique, standardized protocol has to be established. Single-cell qPCR, although partly limited by the number of genes, is relatively simple to analyze. Therefore, its use is accessible without the necessity to recourse to complex bioinformatics analyses. The main steps for single-cell qPCR, as illustrated in this protocol, are composed by single-cell isolation, cell lysate, cDNA reverse-transcription synthesis, amplification for cDNA library generation, and finally, quantitative polymerase chain reaction.

We have developed a protocol to purify RNA from DSS (Dextran Sulfate Sodium)-treated mouse tissues. This method, which prevents downstream inhibition of q-RT-PCR observed in DSS-treated tissues, relies on successive precipitations with lithium chloride.