SAGE (Velculescuetal.1995) is a highly sensitive method based on PCR technology to study gene expression and to obtain qualitative and quantitative information on mRNA libraries. Since the development of this technique in 1995, a large number of articles have been published showing that SAGE can simultaneously detect changes in the expression levels of a large number of genes.
Author: U. Windhorst & H. Johansson Translator: Zhao Zhiqi Chen Jun
Operation method
Series of experiments for analyzing gene expression
Materials and Instruments
Glass and Plasticware Move i. general principles If there is a sufficient amount of RNA for SAGE, preferably 2.5 to 5 ug polyA+RNA, this can be performed as in Experimental Scheme A. The procedure is based on the following principles. Here we also provide protocol B, which has been modified in several steps to be suitable for raw materials with only trace amounts (e.g., using this procedure we were able to obtain gene expression profiles from hippocampal samples derived from 300fxm brain slices). The latter protocol is particularly suitable for studying neural tissue expression, where obtaining large quantities of homogenous tissue for RNA isolation is often impossible due to the complex neural circuits and highly specialized structures. Steps 1 through 8 of both protocols are described in detail below, and from step 9 onwards the steps are the same for both protocols. Many kits isolate PdyA+RNA with good results, and we recommend using the mRNADIRECT kit (Dynal; 610.11) to isolate polyA+RNA from tissues or cultured cells, or the mRNA purification kit (Dynal; 610.01) to isolate pdyA+RNA from total RNA. in the kit instructions All steps are described in detail. 1. TRIzol isolates total RNA (see DD protocol). 2. After RNA precipitation, resuspend 1~10 tons of total RNA in 20 ul lysis buffer (mRNA capture kit). Note: We have had success extracting mRNA with much smaller amounts of total RNA, but this requires some modification in the steps following SAGE. 3. Dilute 20X biotinylated oligo(dT)20 primer (mRNA Capture Kit) to a final concentration of 5 Pmol/ul. 4. Add 4 ul of diluted primer to the lysis buffer containing RNA. 5. Anneal at 37℃ for 5 min. 6. Transfer RNA into a streptavidin-antibiotin-coated PCR tube (mRNA Capture Kit). 7. Incubate at 37°C for 3 min (in this step the mRNA is immobilized on the tube wall by streptavidin-biotin binding). 8. Remove the solution from the tube (containing unbound RNA fragments: rRNA, tRNA, etc.) and carefully rinse the tube 3 times with 50 ul of wash solution (mRNA Capture Kit). 9. Remove the wash solution. 10. Proceed immediately to the cDNA synthesis step. 1. cDNA was synthesized by adding 2.5~5 ug polyA+RNA with oligo(dT)-primer. 2. Mix in a 0.5 ml PCR tube (operate on ice): -2.5 ul polyA+RNA (1 ug/ul) 4 ul 5X first ^ buffer -2 ul 0.lmol/LDTT -1 ul 10 mmol/LdNTPs I 1 ul oligo(dT)18 (biotinylated) (0.5ug/ul) 1 ul SuperScriptHRT (200ug/ul) -8.5 ul DEPC water The total volume was 20 ul. 3. Incubate at 42°C for 2 h (can be done in PCR instrument). 4. Add single-stranded cDNA on ice for second-strand synthesis: (20 ul single-stranded cDNA) One 16 ul 5x second strand buffer -1.6 ul 10 mmol/LdNTPs -2 ul DNA polymerase I (10u/ul) -1 ul T4DNA ligase (5u/ul) -1 ul RNase H (5u/ul) I 38.4 ul redistilled (distilled) water The total volume was 80 ul. 5. Incubate for 2 h at 16°C. 6. Amplify to 200ul with LoTE. 7. Add equal volume of phenol/chloroform/isoamyl alcohol (25:24:1) (PCI). 8. oscillate. 9. Centrifuge at 13OOOr/min for 5 min at 4°C in a microcentrifuge. 10. Transfer the upper aqueous phase to a new 1.5 ml Eppendorf tube. 11. Ethanol precipitation: -3 ul glycogen 100 ul 10 mmol/L ammonium acetate 700 ul Ethanol 12. Place at -20°C for 30 min. 13. Centrifuge at 13OOOr/min for 15 min at 4 "C in a microcentrifuge. 14. Rinse the precipitate twice with 500 ul of 70% ethanol with vigorous shaking. 15. Remove the 70% ethanol and allow the precipitate to air dry for about 15 minutes. 16. Resuspend the precipitate in 20 ul LoTE. Note: The oligo(dT)2a primer (biotinylated, mRNA Capture Kit) used to capture PolyA+RNA and immobilize it to the tube wall (Experimental Scheme B, step 1) can be used directly as a primer for cDNA synthesis, which continues to bind to the wall of the tube after synthesis of the cDNA until it is released by TE digestion in step 6. 1. Rinse the tube in which the PolyA+RNA was captured with 50 ul 1 First Strand Buffer and remove the buffer. 2. Replace IX First Strand Buffer with the following procedure (pipetting on ice): -4 ul of 5X First Chain Buffer 2 ul of 0.1 mol/L TT I ul of 10 mmol/LdNTPs 1 ul of SuperScriptII RT (200 U/ul) -12 ul DEPC water Total volume is 20ul. 3. Incubate for 2 h at 42 °C (on PCR instrument). 4. Remove the reaction mixture from the tubes and wash the tubes once with 50 IX Second Strand Buffer (mRNA Capture Kit). 5. Remove the rinse solution and wash the tube once with 54% IX Second Strand Buffer. 6. Replace the IX first chain buffer with the following solution I 4 ul5X second chain buffer _0.4ul of IOmmol/LdNTPs -Iul DNA polymerase I (10 U/pl) -0.5ul T4DNA Ligase (5 U/-) -0.5ul RNaseH (5U//^l) -13.6 ul Distilled water Total volume is 20ul. 7. Incubate for 2 h at 16℃. Double-stranded cDNA can be stored at -20°C or applied directly in the next step (cDNA digestion with anchoring enzyme). For more product details, please visit Aladdin Scientific website.
Solutions & Buffers Primers
Reagent Kits MPC-E PCR Instrument Gene PulserII System