Table 4 RNA detection methods

RNA fluorescence in situ hybridization (RNA-FISH)

Hybridization signals were observed using fluorescence microscopy after binding to the target RNA with a fluorescent probe complementary to the target RNA sequence

High sensitivity and specificity, multi-color detection, relatively simple and time-consuming operation, tissue morphology can be maintained for detection

High sample requirements, need to ensure RNA integrity, need specialized equipment and probes, high cost, limited accuracy of quantification

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RT-PCR (reverse transcription PCR)

PCR amplification after reverse transcription of RNA to cDNA

It is highly sensitive and specific, suitable for the detection of a wide range of RNAs, less time consuming and more accurate.

Complexity of operation, susceptibility to contamination by foreign products, expensive equipment and reagents

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Northern Blotting

Complexity of operation, susceptibility to contamination by foreign products, expensive equipment and reagents

High sensitivity and specificity, quantitative detection of RNA compared to RNA-FISH

Time-consuming, more complex operations, high sample requirements, need to ensure RNA integrity, need specialized equipment and probes, higher costs

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in situ hybridization

The principle is similar to RNA-FISH, but labeled using markers such as radioisotopes, biotin, digoxin, etc., and finally visualized by radioactive autoradiography, immunohistochemistry, etc.

Both DNA and RNA can be detected at a moderate cost

Not as accurate as RNA-FISH, multiple hybridizations are not as simple as RNA-FISH, can only capture RNA from cells at a certain time point

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RNA microarray

Hybridization of RNA by immobilizing a large number of probes on a microarray

High throughput, accurate quantification and good reproducibility.

Can only detect highly expressed RNAs and cannot cover the full range of RNAs, especially lncRNAs. cost is high and affected by experimental complexity.

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RNA sequencing

Direct sequencing of RNA molecules using high-throughput sequencing technology

Detects all RNAs, capable of deep sequencing with high sensitivity and specificity

Costly, requires advance removal of rRNAs

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