ISH相关技术(—)（ISH related technologies (-)）.docVIP

ISH相关技术(—)（ISH related technologies (-)） (1) kinds of probes; RNA refers to the probe labeled with the organization of the corresponding nucleotide sequence with a complementary single stranded cDNA or cRNA molecules. According to the probe used in RNA hybridization in the source can be divided into three kinds: specific cDNA, cRNA probe and synthetic oligonucleotide probes. 1. single strand cDNA probe: the intron and other highly repetitive sequence does not exist in the cDNA, but also overcome the double stranded cDNA probe in the hybridization reaction between the two chains of complex faults, so as to improve the sensitivity of hybridization reaction. But the single strand cDNA probe preparation is difficult in RNA in situ hybridization has been very there are few applications. 2. cRNA probe: obtained by in vitro transcription using cDNA as template, because it is a single strand probe, therefore To avoid the influence of hybrid hybrid formation between.CRNA and RNA complex problems between the application of two double stranded cDNA probe hybridization reaction to form the ratio between cDNA-RNA stable hybrid.CRNA-RNA by RNA enzyme. Therefore the hybridization reaction available after RNA enzyme treatment, to remove unbound probes because. The cRNA probe has these advantages, cRNA probe hybridization saturation level than the double stranded DNA probe was 8 times higher, so it is widely used in.CRNA probe in situ hybridization in the disadvantage is that the probe preparation process is more complex, need to molecular biology experimental equipment better, it is sensitive to RNA enzyme, easy to be damaged, beware RNA enzyme pollution to operation. 3. oligonucleotide probes: oligonucleotide probes synthesized with nucleotide as raw material, through the synthetic DNA, to avoid the adverse effects of repetitive sequences exist in eukaryotic cells. Since most oligonucleotide sequences bring short, does not require purification, excellent tissue penetration. Specif