RNA radiolabeling

Summary

Intramolecular labeling of RNA during transcription, post-transcriptional labeling of the 5' end of RNA using T4 polynucleotide kinase and labeling of the 3' end of RNA using T4RNA ligase.

Operation method

RNA radiolabeling

Materials and Instruments

[α32P]UTP or CTP (800Ci ml 10mCi ml) UTP or CTP T4 polynucleotide kinase (γ32P)ATP Bovine small intestine phosphatase T4RNA ligase [32P]pCpATP 2Oumol L bovine serum albumin without tRNAase
10XT4 polynucleotide kinase buffer DEPC-treated water 10X phosphatase buffer TE (pH 7.4) 5mol LNaCl TE (pH 7.6) 10XT4RNA ligase buffer DMSO

Move

I Materials and equipment

1) [ ^α32P ]UTP or CTP (800Ci/ml,10mCi/ml).

2) UTP or CTP.

3)T4 polynucleotide kinase

4)10XT4 polynucleotide kinase buffer

5) ( ^γ32P )ATP (3000Ci/mmol; 10mCi/ml)

6) Bovine small intestine phosphatase

7) DEPC-treated water

8) 10X phosphatase buffer

9)TE(pH7.4)

10)5mol/LNaCl

11)TE(pH7.6)

12)T4RNA ligase

13) ^[32P ]pCp (3000Ci/mmol; lOmCi/ml)

14)10XT4RNA Ligase Buffer

15)DMSO

16)ATP, 20umol/L.

17 Bovine serum albumin without tRNAase


II. Methods of operation

(i) RNA internal labeling

1) Add 5ul of [ ^α32P ]UTP or CTP (800Ci/ml, 10mci/ml) to the in vitro transcription system without unlabeled UTP or CTP, or limit their concentration to 10-15umol/L, and incubate for a maximum of 1h.

2) After DNase digestion, purify the labeled full-length RNA by electrophoresis of RNA on denaturing polyacrylamide gel.

(ii) 5' end-labeled RNA

Label the RNA at the 5' end with T4 polynucleotide kinase, which transfers [32P] from [ ^γ-32P ]ATP to the 5' end of the molecule. The 5' end of the labeled RNA molecule requires the removal of the non-isotopic 5' phosphate by bovine small intestine phosphatase.

1) Dilute 1~2ug RNA with DEPC-treated water to a final volume of 16ul, leave at 95℃ for 2 min, then ice bath for 5 min, denature the RNA.

2) Add 2ul of 10X Phosphatase Reaction Buffer, 2ul of Bovine Small Intestine Phosphatase (1U/ul); react for 30 min at 37℃.

3) Dilute the RNA with TE (pH 7.4) to a final volume of 200ul, and add 6ul of 5mol/LNaCl to precipitate the RNA as described above.

4) Resuspend RNA with TE (pH7.6) to a concentration of 0.5ug/ul. Take 2ul of RNA solution, place at 95℃ for 2 min, and then ice bath for 5 min.

5) Add 10XT4 polynucleotide kinase buffer, 4ul of [ ^γ32P ]ATP (3000Ci/mmol, 10mCi/ml), iyT4 polynucleotide kinase (3~6U/ul), incubate at 37°C for 30 min.

6) Purify the labeled RNA by denaturing polyacrylamide gel electrophoresis.

(iii) 3' end-labeled RNA

Label the 3' end of the RNA with T4RNA ligase and an excess of [32P]p4.

1) Place 1~2ug of RNA at 68℃ for 2 min, then ice bath for 5 min, and denature the RNA.

2) Add 4ul of 10XT4 RNA ligase buffer, 4ul of DMSO, 1ul of 20umol/L ATP, 1ul of l0ug/ml RNAase-free bovine serum albumin, 1~2ug of RNA, and 5~10ul of ^[32P ]pCp (3000Ci/mmol, l0mCi/ml), and top up the total volume with DEPC-treated water (40ul). Incubate at 4°C overnight.

3) Purify the labeled RNA by denaturing polyacrylamide gel electrophoresis.

For more product details, please visit Aladdin Scientific website.

https://www.aladdinsci.com/