RNA Detection

suitable for virus packaging, such as the HEK-293FT cell, the resulting lentiviral particles containing the pLenti-d2EGFP- 96mer ...

1 is labeled with a CF640R (Biotium) reporter dye at the 5^0 - end and has the sequence 50 - mCmUmUmCmGmUmCmCmAmCmAmAmAmCmA mCmA ...

2.6 Microscope and Imaging Software An inverted widefield fluorescence microscope. We use an Olympus IX81 motorized inverted fl ...

Transfer 1–1.5 mL of the cell suspension to a microcentrifuge tube. Pellet the cells by centrifugation at 200gfor 5 min at 4C ...

Gently add 350μL wash buffer, incubate for 5 min, and carefully pipette off. Repeat this step once more. After gently removing ...

Apply the preset ROI (step 4) to the filtered stack by selecting it from the ROI manager. The ROI should appear in each slice o ...

4 Notes Due to the multiple tandem repeats, the plasmid should be amplified inEscherichia coliMAX Efficiency Stbl2 (Thermo Fish ...

The number of cells used per microporation is cell type-specific and may affect transfection efficiency and viability. A detail ...

Macros>Install. The plugin can be obtained from:https:// imagej.nih.gov/ij/macros/. In our experience, setting noise toleran ...

Tyagi S, Alsmadi O (2004) Imaging native beta- actin mRNA in motile fibroblasts. Biophys J 87 (6):4153–4162. doi:10.1529/biophy ...

Chapter 16 Optimizing Molecular Beacons for Intracellular Analysis of RNA Mingming Chen, Yantao Yang, Christopher J. Krueger, an ...

by separating the fluorophore from the quencher. This simple yet effective design has led to widespread use of MBs to study the ...

3.2Me/PS10-LOOP. 50 - mGmUmCmAmCmC*mUmC*mAmG*mCmG*mUmA*- mAmG*mUmG*mA*mUmG*mUmC*mGmUmGmAmC-3^0. 4.2Me/PSLOOP 50 - mGmUmCmAmCmC*m ...

Microinjection buffer: 48 mM K 2 HPO 4 , 4.5 mM KH 2 PO 4 , and 14 mM NaH 2 PO 4 , pH 7.2 (seeNote 2). Glass bottom dish (e.g., ...

3 Methods 3.1 Preparation of Reference Dye- Labeled Controls (See Note 5) React 150μL of the aminodextran solution with IRDye® ...

3.3 Cellular Delivery of MB–Dextran Mixtures dextran into a large number of cells with high viability, as shown in our previous ...

Neutralize the remaining trypsin by adding 4.5 mL of cell culture media. Resuspend the cells by pipetting gently. Make sure the ...

Use the “Freehand Selections” tool to draw a Region of Inter- est (ROI) around the object. This object ROI is automatically app ...

Seed HeLa cells microporated with 5μM MBs so that they will be 10–30% confluent at the time of experiment. Dilute the target RN ...

Use a microloader to load the microinjection capillary with 3–5μL of the diluted target RNA solution. This volume is sufficient ...