This experimental program is divided into 6 stages. Stage 1: dephosphorylation of degraded RNA; Stage 2: uncapping of intact RNA; Stage 3: preparation of RNA oligonucleotides; Stage 4: ligation of RNA oligonucleotides to cellular RNA; Stage 5: reverse transcription; Stage 6: amplification. This experiment is from the PCR Laboratory Guide (2nd edition), author: Seed Kang, Qu Lijia.
Operation method
The 5' end of cDNA was amplified by the new RACE method.
Materials and Instruments
Buffers, solutions and reagents Enzymes and enzyme buffers Nucleic acids and oligonucleotides Move Phase 1: Dephosphorylation of degraded RNAs For more product details, please visit Aladdin Scientific website.
Specialty Equipment
I. Materials
1. Buffers, solutions and reagents
Sodium acetate, 3 mol/L, pH 5.2
Ethanol
Dithiothreitol (DTT) (0.1mol/L)
Phenol/chloroform (1:1, v/v)
2. Enzyme and enzyme buffer
Phosphatase buffer, 10X
Protease K
RNasin
Bovine small intestine alkaline phosphatase (CIP)
3. Nucleic acids and oligonucleotides
RNA Sample
4. Specialized equipment
Water bath or heater preset to 37°C, 50°C
5. Other
Reagents and equipment required for agarose gel electrophoresis, including ethidium bromide
II. Methods
In general, phosphatase should be used according to the manufacturer's instructions.
1. Mix the following ingredients in a sterile centrifuge tube.
RNA 50ug
10X phosphatase buffer 5ul
DTT(100 mmol/L) 0.5ul
RNasin(40U/ul) 1.25ul
CIP(lU/ul) 3.5ul
H2O to 50ul
2.50°C incubation for 1h.
3. Add proteinase K to 50ug/ml and incubate at 37°C for 30 min.
4. Extract the reaction with phenol/chloroform and then again with chloroform. Precipitate RNA with l/l0 volume of 3mol/L sodium acetate (pH5.2) and 2.5 times volume of anhydrous ethanol, and re-solubilize RNA with 43. 6ulH20.
5. Take 2ug (1.6ul) of RNA and run 1% agarose gel electrophoresis (TAE) with adjacent lanes spotted with 2ug of the original RNA sample and stain with ethidium bromide to confirm that the RNA is still intact in the dephosphorylation step.
Phase 2: Intact RNA Decap
I. Materials
1. Buffers, solutions and reagents
ATP (100 mmol/L)
Chloroform
Sodium acetate, 3 mol/L, pH 5.2
Ethanol
Phenol/chloroform (1:1, v/v)
TE (10 mmol/LTris-HCl, pH 7.5, lmmol/LEDTA, pH 8.0)
2. Enzyme and enzyme buffer
RNasin
Nicotinic acid pyrophosphatase (TAP), 5 U/ul
TAP Buffer, 10X
3. Nucleic acids and oligonucleotides
RNA samples obtained in phase 1
4. special equipment
Water bath or heater preset to 37°C
5. Other
Reagents and equipment required for agarose gel electrophoresis including ethidium bromide
II. Methods
1. Mix the following ingredients in a sterile centrifuge tube.
RNA (obtained from step 5 of phase 1) 38ul
TAP buffer (10X) 5ul
RNasin (40U/ul) 1.25ul
ATP(100 mmol/L) 1ul
TAP(5U/u1) 1ul
H20 to 50ul
2.37°C for 1h, then add 200ul of TE.
3. Extract the reaction with phenol/chloroform and then again with chloroform. Precipitate RNA with 1/10 volume of 3mol/L sodium acetate (pH 5.2) and 2.5x volume of anhydrous ethanol, and re-dissolve RNA with 40ul H20.
4. Take 2ug of RNA and run 1% agarose gel electrophoresis (TAE) with adjacent lanes spotting 2ug of the original RNA sample and stain with ethidium bromide to confirm that the RNA is still intact during the decap step. 
Phase 3: RNA Oligonucleotide Preparation
Select a plasmid that can be linearized approximately lOObp downstream from the T7 or T3 RNA polymerase site of action [see Figure 25-3(b)]. Because primers from the palindromic sequence of the polyclonal site do not work well for PCR, it is preferable to use a plasmid that is cloned into one of the insert fragments at the polyclonal site. One plasmid that has been validated is pBS-SK-GBX-l-3'UTR, which was cloned into the 3' untranslated region of the mouse Gbx-1 (Frohmanetal.1993) gene at the Sst I site of pBS-SK (Stratagene). It can be linearized with SmaI and transcribed with T3RNA polymerase to produce a 132-nucleotide RNA oligonucleotide. All but 17 of the oligonucleotides are from Gbx-1. Note that adenosine is the best "acceptor" for the 3' terminal oligonucleotide to attach to the 5' end of the target sequence if a suitable cleavage site can be found. The primers used for later amplification are all sequences in the 3' untranslated region. sequences and plasmids for the Gbx-1 NRC primers are available from the Frohman lab. Transcription tests are performed to ensure that the various components are working properly before amplifying the reaction system. Oligonucleotides can be stored at -80°C for long periods of time for backup. It is important to synthesize enough oligonucleotides because of the high losses associated with purification and spot check operations.
I. Materials
1. buffers, solutions and reagents
Chloroform
Ethanol
Diethyl ethyl pyrocarbonate (DEPC) Treated H20
rUTP solution, 10 mmol/L
rATP solution, 10 mmol/L
rCTP solution, 10 mmol/L
rGTP solution, 10 mmol/L
DTT (0.lmol/L)
Phenol/chloroform (1:1, v/v)
Na acetate, 3 mol/L, pH 5.2
TE (10 mmol/L Tris-HCl, pH 7.5, lmmol/L LEDTA, pH 8.0)
2. Enzyme and enzyme buffer
Suitable endonucleases and corresponding buffers
Pancreatic DNAase I (without RNAase)
Protease K
DNA-dependent RNA polymerase
RNasin
Transcription Buffer, 5X (supplied by manufacturer)
3 . Nucleic acids and oligonucleotides
Plasmid template DNA
4 . Specialized equipment
Microcon filter of appropriate size (see step 7)
Water bath or heater preset to 37°C
5 . Other
Reagents and equipment for agarose gel electrophoresis, including ethyl bromide
II.
1. Linearize 25ug of plasmid DNA for transcription with a suitable endonuclease and buffer (make sure that RNAase is not present in the plasmid).
2. digest with 50ug/m l of protease K at 37°C for 30 min, then extract twice with phenol/chloroform and again with chloroform. The DNA is collected by standard anhydrous ethanol precipitation.
3. Solubilize the model DNA with 25ul of TE (pH 8.0). 3. Solubilize the template DNA with 25 ul of TE (pH 8.0) at a final concentration of about lug/ ul.
4. At room temperature, mix the following transcription reagents in a sterilized tube in the following order. 
Incubate at 37°C for 1h
5. After transcription, add 0.5ul of DNAase (no RNAase) per 20ul of reaction volume and incubate at 37°C for 10min to remove the DNA template.
6. Take 5ul of the product of the validation or preparative reaction and run a 1% agarose gel electrophoresis (TAE) to detect the oligonucleotide product. There will be a diffuse band at the expected size (or slightly smaller) of the product, except for some trailing above and below the gel.
7. purify the oligonucleotide by extraction with phenol/chloroform and again with chloroform. Rinse 3 times with water and pass through a Microcon (Millipore) filter (pre-soaked with water).
The Microcon30 filter has a cut-off value of 60 nt and the Microcon100 filter has a cut-off value of 300 nt. If the oligonucleotides are less than 100 nt, the MicroconlO filter may be the most appropriate. For larger oligonucleotides, the Micrcon30 filter can be used.
8. Take another appropriate amount of the oligonucleotide sample and run 1% agarose gel electrophoresis (TAE) to check the integrity and concentration of the sample. Dispense and store at -80°C.
Phase 4: Ligation of RNA oligonucleotides to cellular RNA
I. Materials
1. Buffers, solutions and reagents
ATP, 2 mmol/L
2. enzymes and enzyme buffers
Ligation buffer, lX (500 mmol/LTris-HCl, pH 7.9, 100 mmol/MgCl2, 20 mmol/LDTT, 1 mg/ml BSA)
RNasin
T4RNA ligase
3. Nucleic acids and oligonucleotides
RNA oligonucleotides, prepared from stage 3 above
TAP treated and untreated RNA samples
4. Specialized equipment
Microcon100 Filter
Water bath or heater preset to 17°C
5. Other
Reagents and equipment required for agarose gel electrophoresis including ethidium bromide
II. Methods
1. Take two sterile centrifuge tubes, one with TAP-treated cellular RNA and the other with untreated cellular RNA.
Ligation buffer (10X) 3ul
RNasin (40U/ul) 0.75ul
RNA oligonucleotide 4uundefined
TAP-treated (or non-treated) RNA 10ug
ATP (2 mmol/L) 1.5ul
T4RNA Ligase (20 U/ul) 1.5ul
H2O to 30ul
~undefined 3~6mol more than target cell RNA
2.17°C Overnight reaction for 16 h.
3. Purify the ligand product using a Microcon100 filter (wash 3 times with water, rinse the filter with RNAase-free water beforehand); the recovery volume should not exceed 20ul.
4. Take 1/3 of the ligand product and perform 1% TAE agarose clot electrophoresis to check the integrity of the ligated RNA. It should not change much.
Phase 5: Reverse Transcription
I. Materials
1. buffers, solutions and reagents
dNTP solution (containing 4 dNTP, 10 mmol/L each)
DTT, 0.1mol/L
TE(10mmol/LTris-HCl,pH7.5,lmmol/LEDTA,pH8.0)
2. Enzyme and enzyme buffer
Reverse transcription buffer 5X (supplied by manufacturer)
RNAase H
RNasin
SuperscriptII reverse transcriptase (Invitrogen)
3. Nucleic acids and oligonucleotides
Antisense-specific primer (20ng/ul), or six-base random primer (50ng/ul)
RNA for ligated oligonucleotides, prepared from stage 4 above
4. Specialized equipment
Water bath or heater preset to 37°C, 42°C, 50°C, 70°C
II. Methods
1. In a sterile centrifuge tube, mix the following transcription components on an ice bath.
Reverse transcription buffer, 5X 4ul
dNTP solution (10 mmol/L) 1ul
DTT (0.lmol/L) 2ul
RNasin (40U/ul) 0.25ul
2. In another tube, dissolve the remaining RNA (about 6.7ug) in 13ul of water, add 20ng of antisense-specific primer or 50ng of hexabase random primer, incubate at 80°C for 3 min, cool rapidly on ice, and centrifuge for 5s.
3. add the RNA/primer mixture to the reverse transcription fraction, then add 1ul (200U) of SuperscriptII Reverse Transcriptase, incubate at 42°C for lh, then at 50°C for 10 min. if random primers are used, after mixing the fractions, add one step of incubation at room temperature for 10 min.
4. incubate at 70°C for 15 min to inactivate the reverse transcriptase, then centrifuge for 5 s. If a random primer is used, add a 10-min incubation step at room temperature after mixing the components.
5. Add 0.75ul (1.5U) of RNAase H and incubate at 37°C for 20 min to disrupt the RNA template.
6. Dilute the reaction mixture to 100ul with TE (10 mmol/L Tris-HCl, pH 7.5, lmmol/LEDTA) and store at 4°C (this is the 5' end oligo-cDNA library).
Phase 6: Amplification
i. Materials
1. buffers, solutions and reagents
TE (10 mmol/LTris-HCl, pH 7.5, lmmol/LEDTA, pH 8.0)
DMSO
2. Enzyme and enzyme buffer
dNTP solution (contains 4 dNTPs, 10 mmol/L each)
Taq polymerase (missing from the original. -Add by translator)
Taq polymerase buffer (10X)
3. Nucleic acids and oligonucleotides
5' terminal oligo-cDNA library (from step 6 of phase 5)
Oligonucleotide primers GSP1, GSP2, NRC1 and NRC2 (see Figure 25-4 for details of NRC1 and NRC2 primers)
4. Specialized equipment
Programmable Thermal Cycler
Methods
1. First round
(1) Mix the following components in a sterile 0.2 ml centrifuge tube.
Taq Polymerase Buffer (10X) 5ul
dNTP solution (10 mmol/L) 1.0ul
DMSO 5ul
Tag polymerase 2.5U (missing in original. --Translator's addition)
H20 Add to 50ul
(2) Take 1ul of 5'-end oligo-cDNA library and add 25pmol each of GSP1 and NRC1 primers.
(3) Heat at 98°C for 5 min on a DNA thermal cycler to denature the first strand product and activate the polymerase. Cool to the appropriate temperature (56?68°C), anneal for 2 min, and extend at 72°C for 40 min.
(4) Perform 30 (originally 35) amplification cycles according to the following program. The following procedure was followed for 30 cycles of amplification. 
2. Second cycle
(5) Take a portion of the amplification product from the first round and dilute it 1:20 with TE.
(6) Using the procedure described above, but omitting the 2 min annealing and 40 min extension at 72°C, amplify a 1ul dilution of the sample with GSP2 and NRC2 primers. 