PAT method for analyzing mRNA Poly(A) tail length assay
The PAT method [ PCR poly(A) test ] can quickly and sensitively analyze specific mRNA from total RNA and quantitatively estimate the length of RNA poly(A) tails. In this method, the entire process, including cDNA synthesis, is performed in a single reaction tube, which not only simplifies the operation, but also reduces the possibility of RNase contamination. This experiment is from "RNA Laboratory Guidebook", edited by Xiaofei Zheng.
Operation method
Scheme 5.10 Analysis of mRNA Poly(A) tail length by PAT method
Principle
The PAT method [ PCR poly(A) test ] can quickly and sensitively analyze specific mRNA from total RNA and quantitatively estimate the length of RNA poly(A) tails. In this method, the whole process including cDNA synthesis is done in one reaction tube, which not only makes the operation easier, but also reduces the possible RNase contamination. In particular, the PAT cDNA is very stable and reusable after synthesis, and the ease of use and sensitivity of this method make it possible to analyze poly(A) tail lengths in any system, and it has been successfully applied to the analysis of poly(A) in murine oocytes, Drosophila oocytes and liver foetuses, nematodes, yeast and cultured cells.
Materials and Instruments
T4 DNA ligase None RNase H Superscript II reverse transcriptase Oligomeric (dT) anchor primer mRNA-specific primer Move I. Materials and equipment For more product details, please visit Aladdin Scientific website.
Distilled water Superscript II RT buffer DTT ATP dNTP Taq DNA polymerase Taq DNA polymerase buffer
1. PAT cDNA preparation
(1) DEPC treated distilled water.
(2) High concentration of T4 DNA ligase (10 Weiss U/L).
(3) RNase H Superscript II-free reverse transcriptase (RT) ( Life Technologies, Gairhersburg, MD, USA) ( 200 U/μl ).
(4) 5 X Superscript II RT buffer.
(5) 0.1 mol/L DTT, DEPC water preparation.
(6) 10 mmol/L ATP in DEPC water.
(7) 40 mmoI/L dNTP (10 mmol/L dATP, 10 mmol/L dTTP, 10 mmol/L dCTP, 10 mmol/L dGTP), DEPO-H2O preparation.
(8) p[dT ]12-18 Note that oligo[dT ]12-18 must be phosphorylated ], 10 ng/μl.
(9) Oligo (dT ) anchor primer (5'-GCG AGC TCC GCG GCC GCG T12 ) 200 ng/μl (other oligo with oligo dT extension at 3' end (n>10) and 5' GC-rich oligonucleotide can also be used as PCR primer), DEPC water preparation, all the above enzymes and solutions were stored at -20℃.
2. PCR reaction
(1) 40 mmol/L dNTP (10 mmol/L dATP, 10 mmol/L dTTP, 10 mmol/L dCTP, 10 mmol/L dGTP) in DEPC water.
(2) Oligomeric (dT ) anchor primer 200 ng/μl water.
(3) mRNA-specific primers, dissolved in distilled water.
(4) Taq DNA polymerase.
(5) 10X Taq DNA polymerase buffer.
II. Methods of operation
1. PAT cDNA preparation
(1) Extract total RNA from tissues or cells, dissolve in DEPC water and add 5 μl into a microcentrifuge tube without RNase.
(2) Add 2 μl (20 ng) of p[dT ]12-18 to each RNA sample, and the total volume of RNA and p[dT ]12-18 is 7 μl.
(3) Heat to 65°C and denature for 5~10 min.
(4) Immediately place the reaction tube in a 42°C water bath, taking care to move quickly to avoid annealing of p[dT ]12-18 with the least poly(A) tail.
(5) Add 13 μl of the following mixture preheated to 42°C, mix well by pipetting, and incubate at 42°C for 30 min. For all PAT reactions, pre-mix all the same components, and for each reaction, add: 4 μl of 5X Superscript II RT buffer, 2 μl of 0.1 mol/L DTT, 1 μl of 40 mmol/L dNTP mixture, 1 μl of 10 mmol/L dNTP mixture, and 1 μl of 10 mmol/L dNTP mixture. mixture, 1 μl 10 mmol/L ATP, 4 μl DEPC-treated water, 1 μl T4 DNA Ligase 10 Weiss U/μl.
(6) After incubation, the counterstain was kept at 42°C and 1 μl (200 ng) of oligo(dT) anchor primer was added. Mix well, centrifuge quickly, and incubate at 12°C for 2 h. Incubate for 2 h at 12°C.
(7) Incubate the reaction tube at 42 ℃ for 2 min.
(8) Add 1 μl of RNase H Superscript II-free reverse transcriptase, mix well, and incubate at 42°C for 1 h. Incubate for 2 h at 12°C for 10 min.
(9) Heat for 20 min to inactivate the reverse transcriptase and ligase, and the PAT cDNA can be diluted as needed.
2. PCR reaction
(1) 25~50 μl standard reaction system includes: 1X buffer [ 50 mmol/L KCl, 10 mol/L Tris-HCl (pH 9.0 at 25°C), 0.1% Triton X-100, 1.5 mmol/L MgCl2 ], 0.2 mmol/L dNTP, 0.5 μmol/L template specific primer, 0.5 μmol/L oligo (dT) anchor primer, and 0.5 μmol/L template specific primer. μmol/L oligo(dT) anchor primer, 0.5~1.0 U Taq DMA polymerase, 0.5~1.0 μl PAT cDNA as template.
(2) Suggested cycling conditions for amplification of 500 bp fragment: denaturation at 93°C for 5 min, variation at 93°C for 30 s, repeat at 57-65°C for 1 min, extension at 72°C for 1 min, and finally extension at 72°C for 7 min.
(3) A small portion of the amplification product can be taken and analyzed by complete digestion of the 3' end using restriction endonuclease.
(4) Analyze the amplified product by gel electrophoresis.