Assaying of nucleic acid Experiment 12 Assaying of nucleic acid

Aim and request Comprehend the principle and method of the mensuration of DNA and RNA.

Principle There are two types of nucleinic acid: DNA and RNA. DNA chiefly reside in cellular nucleus，and in cytoplasm RNA is most plentiful. In alkaline solution RNA can be hydrolyzed, its ribose dehydrated to form furfurol, then furfurol can Kondens with orcinol to synthesize green compound, the shade of green is in direct proportion to the amount of RNA.

RNA hydrolyze ribose OH- dehydrate furfurol orcinol green compound

Heated in perchloric acid solution, DNA can be hydrolysed, its ribodesose dehydrated to formω- hydroxy-γ-keto- pentanal, then it can react with diphenylamine to produce blue compound, which chemical property is not clear, but the shade of blue is in direct proportion to the amount of DNA.

DNA ribodesose ω- hydroxy-γ-keto - pentanal ribodesose diphenylamine hydrolyze DNA ribodesose perchloric acid dehydrate ω- hydroxy-γ-keto - pentanal ribodesose diphenylamine blue compound

Procedure ⑴ RNA assaying ①alkaline hydrolysis：Take two centrifuge tubes, put cytoplasm and 1mol/L KOH 1ml each in the tube1; put nucleochylema and 1mol/L KOH 1ml each in the tube2. After mix, put the two tubes in the boiling water for 10min, after cool down, add 0.3mol/L trichloracetic acid 4ml in each tube, mix it, then centrifugate at 3000rpm for 5min. The supernatant is the alkaline digest of RNA.

Tube2 Tube 1 Cytoplasm 1ml KOH 1ml nucleochylema 1ml 100℃water bath, 10min Cool down trichloracetic acid 4ml Centrifugate, 3000rpm for 5min the alkaline digest of RNA

②Coloration and assaying: Take three tubes, add reagents according to table1, and mix immediately, then place them into boiling water for 10min, after cool down, adjust the spectrophotometer to zero with blank tube(tube3). Determine the absorbance of each tube under 660 nm wavelength. Look up in the RNA standard curve to get the content of RNA in cytoplasm and cellular nucleus digest.

reagent(ml) tube number Table 1 RNA assaying cytoplasm digest 1.0 — — nucleonic digest — 2.0 — 0.3mol/L trichloracetic acid 1.0 — 2.0 orcinol 3.0 3.0 3.0 reagent(ml) tube number   Table 1 RNA assaying 1 2 3(blank)

⑵ DNA assaying ① Acid hydrolysis: Take two centrifuge tubes, put cytoplasm and 1mol/L perchloric acid 2ml each in the tube1; put nucleochylema and 1mol/L perchloric acid 2ml each in the tube2. Heat in 95℃ for 10min，after cool down，centrifugate at 3000rpm for 5min. The supernatant is the acid digest of DNA.

Tube 4 Tube 3 Cytoplasm 2ml perchloric acid 2ml nucleochylema 2ml 95℃water bath, 10min Cool down Centrifugate, 3000rpm for 5min the acid digest of DNA

② Coloration and assaying:：Take three tubes, add reagents according to table3, and mix immediately, place them in boiling water for 12min, after cool down, Adjust the spectrophotometer to zero with blank tube (tube3). Determine the absorbance of each tube under 595 nm wavelength. Look up in the DNA standard curve to get the content of DNA in cytoplasm and cellular nucleus digest.

reagent(ml) tube number   Table 3 DNA assaying cytoplasm digest 2.0 — — nucleonic digest — 2.0 — 0.5mol/L perchloric acid — — 2.0 diphenylamine 2.0 2.0 2.0 reagent(ml) tube number 1 2 3

Results and calculation Tube 1 2 3 OD RNA content in cytoplasm of alkaline hydrolysis RNA content in nucleochylema of alkaline hydrolysis DNA content in cytoplasm of acid digest DNA content in nucleochylema of acid digest

⑴ RNA content of the sample（ug/ml）= RNA content of alkaline hydrolysis×dilute multiple (cytoplasm 6 times，nucleochylema 3 times)

⑵ DNA content of the sample (mg/ml)= DNA content of acid digest ×dilute multiple (cytoplasm and nucleochylema both 2 times) ÷usage amount of cytoplasm and nucleochylema (both 2ml) ⑶ calculate the ratio of RNA and DNA in the cytoplasm and nucleochylema.

Application significance Use this method we can quantitative determination the genetic material such as RNA or DNA of the cells, thus provide a necessary supplementary method for separation and mensuration technique about nucleic acid in clinical research.

Cautions ⑴after centrifugation, imbibing supernatant, must avoid sucking in sediment as far as possible, otherwise causing relative accuracy of the result. ⑵ When calculate the RNA or DNA content, must pay attention to the dilute multiple of the sample. ⑶ When determine the absorbance, must pay attention to that the wavelength of RNA and DNA is different.