Journal of biological methods最新文献

The preparation of small RNA cDNA sequencing libraries depends on the unbiased ligation of adapters to the RNA ends. Small RNA with 5' recessed ends are poor substrates for enzymatic adapter ligation, but this 5' adapter ligation problem can go undetected if the library preparation steps are not monitored. Here we illustrate the severity of the 5' RNA end ligation problem using several pre-miRNA-like hairpins that allow us to expand the definition of the problem to include 5' ends close to a hairpin stem, whether recessed or in a short extension. The ribosome profiling method can avoid a difficult 5' adapter ligation, but the enzyme typically used to circularize the cDNA has been reported to be biased, calling into question the benefit of this workaround. Using the TS2126 RNA ligase 1 (a.k.a. CircLigase) as the circularizing enzyme, we devised a bias test for the circularization of first strand cDNA. All possible dinucleotides were circle-ligated with similar efficiency. To re-linearize the first strand cDNA in the ribosome profiling approach, we introduce an improved method wherein a single ribonucleotide is placed between the sequencing primer binding sites in the reverse transcriptase primer, which later serves as the point of re-linearization by RNase A. We incorporate this step into the ribosomal profiling method and describe a complete improved library preparation method, Coligo-seq, for the sequencing of small RNA with secondary structure close to the 5' end. This method accepts a variety of 5' modified RNA, including 5' monophosphorylated RNA, as demonstrated by the construction of a HeLa cell microRNA cDNA library.

Multi-color flow cytometry is a standard laboratory protocol, which is regularly used to analyze tumor-infiltrating immune cell subsets. Oncolytic herpes simplex virus has shown promise in treating various types of cancers, including deadly glioblastoma. Intracranial/intratumoral treatment with oncolytic herpes simplex virus expressing interleukin 12, i.e., immunovirotherapy results in induction of anti-tumor immune responses and tumor infiltration of a variety of immune cells. Multi-color flow cytometry is employed to characterize immune cells in the tumor microenvironment. Here, we describe a step-by-step 11-color flow cytometry protocol to stain tumor-infiltrating immune cells in glioblastoma following oncolytic herpes virotherapy. We also describe a method to identify HSV-1 glycoprotein-B-specific CD8⁺ T cells using fluorochrome-conjugated major histocompatibility complex multimers. The multimers carry major histocompatibility peptide complexes, which have the ability to interact and bind to T cell receptors present on the surface of T cells; allowing identification of T cells (e.g., CD8⁺) reactive to a desired antigen. This multimer staining can be used in conjunction with the multi-parametric flow cytometry staining. Brain tumor quadrants are harvested, minced, enzymatically digested, immune cells are isolated by positive selection, single cells are counted and blocked for Fc receptors, cells are incubated with dye and/or color-conjugated antibodies, and flow cytrometry is performed using a BD LSRII flow cytometer. The protocol described herein is also applicable to stain immune cells in other mouse and human tumors or in any desired tissues.

We present a new approach to quantify the half-life of membrane proteins on the cell surface, through tagging the protein with the photoconvertible fluorescent protein, Dendra2. Upon exposure to 405 nm light, Dendra2 is photoconverted from green to red emission. Total internal reflection fluorescence microscopy (TIRF) is applied to limit visualization of fluorescence to proteins located on the plasma membrane. Conversion of Dendra2 works as a pulse chase experiment through monitoring only the population of protein that has been photoconverted. As the protein is endocytosed the red emission decreases due to the protein leaving the TIRF field of view. This method is not impacted by the insertion of new protein into the plasma membrane as newly synthesized protein only exhibits green emission. We used this approach to determine the half-life of ENaC on the plasma membrane illustrating the high temporal resolution capability of this technique compared to current methods.