In Situ Hybridization
It involves the hybridization of a labeled probe to a complementary target sequence, allowing for the visualization and analysis of gene expression patterns, chromosome structure, and viral infections.
ISH has numerous applications in basic research, diagnostic medicine, and drug discovery, enabling researchers to gain insights into cellular and molecular processes.
This versatile method can be applied to a wide range of sample types, including cultured cells, tissue sections, and whole organisms.
With its ability to provide spatial and temporal information about target molecules, in situ hybridization has become an indispensable tool in the field of molecular biology and genetics.
Most cited protocols related to «In Situ Hybridization»
For each gene, the voxel data is a 67 × 41 × 58 (rows × columns × depth) array, giving an “energy” value representing the expression. In addition, for each voxel we know the anatomical annotation. The Allen Brain reference atlas is given at a finer resolution with voxels of 25μm (528 × 320 × 456). In order to achieve finer resolution and smoother images we used linear interpolation of the coarse (200μm) in situ data into the finer grid (25μm). For annotation we used the color code of the Allen reference atlas.
Since many genes have information only from sagittal sections of one hemisphere, we can neglect one hemisphere also from the genes that have coronal data. Coronal data is preferred since it has better sampling.
Most recents protocols related to «In Situ Hybridization»
Example 4
The ability of certain, active HAO1-targeting DsiRNAs to reduce HAO1 levels within the liver of a mouse was examined. DsiRNAs employed in the study were: HAO1-1105, HAO1-1171, HAO1-1221, HAO1-1272, HAO1-1273, HAO1-1316, HAO1-1378 and HAO1-1379, each of which were synthesized with passenger (sense) strand modification pattern “SM107” and guide (antisense) strand modification pattern “M48” (patterns described above). To perform the study, a primary hyperoxaluria model was generated through oral gavage of 0.25 mL of 0.5 M glycolate to cause urine oxalate accumulation in C57BL/6 female mice. Animals were randomized and assigned to groups based on body weight. Intravenous dosing of animals with lipid nanoparticles (LNPs; here, an LNP formulation named EnCore-2345 was employed) containing 1 mg/kg or 0.1 mg/kg of DsiRNA was initiated on day 0. Dosing continued BIW for a total of three doses in mice prior to glycolate challenge. Four hour and 24 h urine samples were collected after glycolate challenge for assessment of oxalate/creatinine levels (see
Robust levels of HAO1 mRNA knockdown were observed in liver tissue of mice treated with 1 mg/kg amounts of HAO1-targeting DsiRNAs HAO1-1171 and HAO1-1378 (
In additional in vivo experiments, both HAO1 and oxalate levels were assessed in both control- and DsiRNA-treated genetically engineered PH1 model mice.
Example 10
LacZ and Luciferase Modified RNA Cardiac Transfection and Translation in a Citrate Saline Buffer
As depicted, 75 μg of LacZ encoding modified RNA with cardiac injections was transfected and translated in approximately 10% of the left ventricle (
Animals
Animal No | Total body length (cm) | Body weight (g) | Sex | Application | |
---|---|---|---|---|---|
P. aethiopicus | 1 | 50.0 | 349.0 | F | ISH (left)/RNA extraction (right) |
2 | 35.0 | 150.6 | M | Dice CT | |
3 | 31.5 | 100.0 | unknown | ISH | |
4 | 34.0 | 118.3 | F | SEM | |
L. paradoxa | 1 | 65.0 | 994.5 | F | RNA extraction (left)/ISH (right) |
3 | 18.5 | 18.6 | M | ISH |
ISH in situ hybridization; Dice CT Diffusible iodine-based contrast-enhanced computed tomography; SEM Scanning Electron Microscopy
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More about "In Situ Hybridization"
This powerful method involves the hybridization of a labeled probe, such as those created using DIG RNA Labeling Kit or DIG RNA labeling mix, to a complementary target sequence.
The visualization and analysis of the hybridized probe, often facilitated by the use of NBT/BCIP or BM Purple, enables researchers to study gene expression patterns, chromosome structure, and viral infections.
ISH has a wide range of applications in basic research, diagnostic medicine, and drug discovery, making it an indispensable tool in the field of molecular biology and genetics.
The technique can be applied to a variety of sample types, including cultured cells, tissue sections, and whole organisms.
To ensure optimal results, researchers may utilize Superfrost Plus slides and Alkaline phosphatase-conjugated anti-DIG antibody to enhance the signal detection and visualization.
The versatility of ISH is further enhanced by the use of plasmid vectors, such as PGEM-T Easy, which can be used to generate the labeled probes using T7 RNA polymerase.
Optimizing ISH protocols can be a time-consuming and challenging task.
PubCompare.ai, an AI-driven optimization tool, can help researchers locate the best protocols from literature, pre-prints, and patents, using intelligent comparisons to identify the most effective methods and products.
This can improve experimental reproducibility and save valuable time, making the in situ hybridization process more efficient and effective.