Microbiology

The current study provides detailed protocols utilized to amplify the complete HIV-1 gp120 and nef genes from single copies of expressed or integrated HIV present in fresh-frozen autopsy tissues of patients who died while on combined antiretroviral therapy (cART) with no detectable plasma viral load (pVL) at death (Lamers et al., 2016a and 2016b; Rose et al., 2016). This method optimizes protocols from previous publications (Palmer et al., 2005; Norström et al., 2012; Lamers et al., 2015; 2016a and 2016b; Rife et al., 2016) to produce single distinct PCR products that can be directly sequenced and includes several cost-saving and time-efficient modifications.

Studying the transcriptome of bacterial pathogens during infection is a very informative and effective tool for discovering genes that contribute to successful infection. However, isolating bacterial RNA from infected cells or tissues is a challenging process due to the much higher amounts of host RNA in the lysates of infected cells. We have optimized a method for isolating RNA of Listeria monocytogenes (L. monocytogenes) bacteria infecting bone marrow derived macrophage cells (BMDM). After infection, we lyse the cells and filter the lysates through 0.45 µm filters to discard most of the host proteins and RNA. Next, we resuspend the bacteria and extract RNA following DNase treatment. The extracted RNA is suitable for gene expression analysis by real-time PCR or microarray. We have successfully employed this protocol in our studies of Listeria monocytogenes gene regulation during infection in vitro (Lobel et al., 2015; Lobel et al., 2012; Kaplan Zeevi et al., 2013; Rabinovich et al., 2012).

The protocol describes the procedure of total RNA isolation from cells of the cyanobacterium Synechocystis sp. PCC 6803. This protocol is also applicable to Synechococcus elongatus PCC 7942 and PCC 6301, Thermosynechococcus vulcanus, and other unicellular and filamentous species of cyanobacteria that do not have thick polysaccharide-containing outer layers. For the latter, Trizol-containing protocols should be adapted. The yield of RNA depends on optical density of cyanobacterial culture and may reach up to 10-20 µg of total RNA per 1 ml of cell culture. RNA isolated by this method can be used for Northern blot hybridization, RT-qPCR, microarrays and Next Generation Sequencing.

The northern blot is a technique used in molecular biology research to study gene expression by detection of RNA in a sample. With northern blotting it is possible to observe particular gene expression levels during differentiation, morphogenesis, as well as abnormal or diseased conditions. Here, we examine ATF3, ATF4, and GADD153 gene expression profiles by northern blot in Vero cells and H1299 cells after IBV infection. RNA was extracted in IBV (infectious bronchitis virus) infected cells and electrophoresis was used to separate the RNA sample. RNA was transferred from the electrophoresis gel to the blotting membrane by capillary transfer. Specific mRNA was detected with hybridization probes complementary to part of target sequence. The probes were prepared by RT-PCR and labeled by digoxigenin (DIG) using DIG labeling kit.

Next generation sequencing has allowed for the analysis and ability to identify the microbial communities present in the environment. While DNA extraction from environments (such as soil) have provided a wealth of knowledge regarding microbial communities there are drawbacks that one encounters when using DNA as opposed to RNA. RNA allows for the determination of the identity of the microbes that are active and present at a particular time point and thus gives a clear picture of what these microbes are actually doing at a specific point in time and under a set of conditions. Extracting RNA from soil is challenging due to the inherent inhibitors present in the soil such as humic acids. Here we describe modifications to the MoBio RNA PowerSoil^TM total RNA isolation kit to reproducively extract total RNA from the soil.

Compared to several expensive RNA extraction kits, the following protocol provides an economic and simple method for researchers to extract yeast RNA. This method can achieve RNA quality that is sufficient for most northern blot studies in yeast.