clicks:9
2022-06-09 10:05:14
Northern Hybridization

One. Experimental Principle

Northern hybridization uses agarose gel electrophoresis to separate RNAs with different molecular weights, and then transfers them in situ to solid-phase supports (such as nylon membrane, nitric acid fiber membrane, etc.), then hybridizes with radioactive (or non radioactive) labeled DNA or RNA probes according to their homology, and finally performs autoradiography (or chemical development), indicating the molecular weight of the target RNA by its location, The development intensity can indicate the relative content of target RNA in the measured sample (i.e. the abundance of target RNA).

Two. Application Introdcution

1. Qualitative and quantitative analysis of gene transcription level differences;

2. Detect whether the target gene has variable shear products or repeats.

Three. Experimental Methods

image.png

Four. Sample Delivering Requirements

Sample type

Sample requirements

Preservation conditions

Delivery conditions

Note

Animal tissue

A single sample should be  less than 0.1g, and installed in 2ml EP tube or frozen storage tube, and do not overdose it; Samples should be as fresh as possible. If protein or nucleic acid cannot be extracted immediately, they should be frozen at - 80 ℃ or lower after quick freezing with liquid nitrogen. Frozen samples should avoid repeated freezing and thawing to avoid degradation.

At - 80 ℃

With dry ice

All samples need to be uniquely marked and the markings are clearly identifiable

Seed sample

A single sample of seed that are shelled and fresh or stored in liquid nitrogen should be less than 0.2g

At - 80 ℃

With dry ice

Adherent/Suspension cell

1. Total RNA or protein: 10^5 cell/index, plasma/ nuclear RNA or protein:10^7 cell/index, mitochondrial RNA or protein: 2*10^7 cell/index.the samples should be as fresh as possible and should directly add Trizol (QPCR test) or frozen at  - 80 ℃ after collected.

2. If the cells are in poor condition after treatment (dosing, transfection and infection), the sample collection volume should be increased as appropriate

At - 80 ℃

With dry ice

Whole blood/serum samples

5 to 10 ml of peripheral blood and 1 to 3 ml of bone marrow preserved with anticoagulant tubes. The leukocyte homogenate stored at -80 ℃ for no more than half a week is less than 400 μ L, and 400 μ L Trizol is added every 400 μ L

At - 80 ℃

With dry ice

Paraffin embedded samples

The effective thickness of paraffin block embedded by standard paraffin embedding box should be thicker than 0.1cm; The thickness of each fresh FFPE tissue sections should be no more than 10 microns, and a surface area of no more than 250 mm^2, 2 to 8 piece in total.

At - 20

With ice bag

Antibody

1. Provide antibodies that meet the corresponding experimental requirements according to the sample species;

2. Send according to the requirements of the antibody manual, and try to avoid sub packaging; In case of sub packaging, ensure that the amount is sufficient for the experiment, and provide the antibody instruction;

3. The antibody tube storing the antibody should have a mark that can recognize the antibody, and the amount of antibody should be greater than the amount required for the experiment.

At - 20

With ice bag

Primers

The primers should be dry powder and less than or equal to 1 OD. If pre experiment is conducted, at least two pairs of primers should be provided for each gene, and the primer synthesis sheet should be attached by the company,

At - 20

With ice bag or at ambient temperature 

Five. Case Display

image.png

Six. Common Problems

1. RNA is easy to be degraded by RNases in the environment, so we must pay special attention to the pollution of RNases. The operation must be carried out carefully and in strict accordance with the isotope operation procedures to prevent isotope pollution.

2. If necessary, the labeled probe can be purified by sephades G-50 column chromatography to remove unbound (free) nucleotides in the labeling reaction.

Seven. Service Process

image.png