RACE amplification
6/26/00 AK
Preparation and handling of poly A+ RNA
1. General precautionsThe integrity and purity of your poly A+ RNA used as starting material is an important element of high-quality cDNA synthesis. The following precautions will help you avoid contamination and degradation of your RNA:
a. Wear gloves.
b. Use freshly deionized (e.g" MilliQ-grade) H20 directly, without treatment with DEPC (diethyl pyrocarbonate).
c. Rinse all glassware with 0.5 N NaOH, followed by deionized H20.Then bake the glassware at 160-180 oC for 4-9 hr.
d. Use only single-use plastic pipettes and pipette tips with RNA.
2. RNA isolation
Many procedures are available for the isolation of polyA+ RNA (Farre[i,
1993; Sambrook etaI., 1989). I use direct mRNA purification kit using oligotex from QIAGEN.
1. From 32 hr embryos of ~100 ul volume, add 1000 ul OL1+ 30 ul b-ME in 3 microtubes.
2. Add 670 ul of Dilution buffer to each tube.
3. Centrifuge 3min
4. Add oligotex suspension 33 ul per tube. RT for 10 min.
5. Centrifuge 5 min
6. Suspend in 350 ul Wash buffer OW1. Pipet into spin column.
7. Centrifuge 30 sec
8. Wash 2 x 350 ul OW2
9. Elute 2 x 100 ul Elution buffer at 70 oC
10. Precipitate mRNA (~10 ug mRNA, checked on agarose gel)
11. Dissolved in 10 ul H2O
(alternative ) You can use total RNA instead of mRNA
I usually use TRIzol RNA isolation solution (GIBCO BRL). Use ~20 x volume of solution to the number of embryo (ex. for 10 embryo use 200 ul). I usually obtain ~1 ug total RNA from one ZF embryo.
3. RNA analysisAfter isolating poly A+ RNA, we recommend that you examine the RNA by agarose gel. Poly A+ RNA samples should produce smears from 0.5-12 kb with much weaker ribosomal RNA bands at approximately 4.5 and l.9 kb. Size distribution may be smaller with non-mammalian tissue sources.
First-strand cDNA synthesis
Use Clontech Marathon cDNA amplification kit to get missing 5’ and 3’ fragments. The 10-ul reaction described below converts l ug of poly A+ RNA into first-strand cDNA. Following second-strand synthesis, the ds cDNA is ready for ligation to the cDNA Adaptor. The kit contains enough components for five separate cDNA syntheses. We strongly recommend that you perform a positive control cDNA synthesis with the human placental poly A+ RNA that is included in the kit. This will verify that the system performs in your hands and will allow you to estimate the yield and size distribution of the ds cDNA synthesized from your RNA. This information is very important for successful amplification of full-sized 5' & 3’ cDNA fragments. The ds cDNA you make from the positive control RNA will - together with the TFR primers - provide a positive control for the 5'- and 3'-RACE PCR reactions.
l. Combine the following in a sterile 0.5-ml microcentrifuge tube:1 ul poly A+ mRNA
1 ul cDNA synthesis primer (10 uM)
3 ul H2O2. Mix contents and spin the tube briefly in a microcentrifuge.
3. Incubate the tube at 70 oC for 2 min.
4. Cool the tube on ice for 2 min.
5. Spin the tube briefly to collect the contents at the bottom.
6. Add the following to each reaction tube:
2 ul 5X first-strand buffer
l ul dNTP mix (10mM)
l ul [a-32p] dCTP (1 uCi/ul) I
l ul MMLV reverse transcriptase (100 units/ul)
10 ul Total Volume
7. Mix the contents of the tube by gently pipeting.8. Spin the tube briefly to collect the contents at the bottom.
9. Incubate the tube at 42 oC for l hr in an air incubator.
Note: Using a water bath or the rmocycler for this incubation may reduce the volume of the reaction mixture (due to evaporation) and therefore reduce the efficiency of first-strand synthesis.
l 0. Place the tube on ice to terminate first strand synthesis.
11. Proceed directly to 2nd strand synthesis.
Second-strand cDNA synthesis
This procedure describes an 80-ul reaction in which second-strand cDNA is synthesized from the first-strand cDNA produced in the 10-ul first-strand reaction. The second-strand enzyme cocktail contains RNase H, E. coli DNA polymerase I, and E. coli DNA ligase. These enzymes degrade the RNA and synthesize the second cDNA strand. The action of T4 DNA polymerase creates blunt ends on the ds cDNA.
We recommend that you also perform a positive control second-strand synthesis using the first-strand cDNA made from the human placental poly A+ RNA provided with the kit. This is necessary if you want to have a positive control for later steps.
Note: All components and reaction vessels should be pre-chilled on ice.
1. Combine the foIllowing components in the reaction tube:
(10 ul First-strand reaction volume)
48.4 ul Sterile H20
16 ul 5 x Second-strand buffer
l.6 ull dNTPmix(10mM)
4 ul 20 x second-strand enzyme cocktail
80 ul Totalvolume
2. Mix contents thoroughly with gentle pipeting.3. Spin the tube briefly to collect the contents at the bottom
4. Incubate the tube at 16 oC for l.5 hr.
5. Add 2 ul (10 units) of T4 DNA po[ymerase and mix thoroughly with gentle pipeting.
6. Incubate the tube at 16 oC for 45 min.
7. Add 4 ul of the EDTA/GIycogen mixture to terminate second-strand synthesis.
8. Add l00 ul of phenol:chloroform:isoamyl alcohol (25:24:l).
9. Vortex thoroughly.
10. Spin the tube in a microcentrifuge at 14,000 rpm for 10 min to separate phases.
l l. Carefully transfer the top aqueous layer to a clean 0.5-ml microcentrifuge tube. Discard the interface and lower phase.
12. Add l00 ul of chloroform:isoamyT alcohol (24:l) to the aqueous layer and vortex thoroughly.
13. Spin the tube in a microcentrifuge at 14,000 rpm for 10 min to separate phases.
14.Remove the top aqueous layer and place in a clean 0.5-ml microcentrifuge .
15. Add one-half volume of 4M ammonium acetate. (e.g" if you recovered = 70 ul at Step 14, add 35 ul of4 M ammonium acetate)
16. Add 2.5 volumes of room-temperature 95% ethanol. (e.g., if your olume at Step 15 was = 105 ul, add 263 ul of 95% ethanol)
17. Vortex the mixture thoroughly.
18. Spin the tube immediately in a microcentrifuge at 14,000 rpm at room temperature for 20 min.
Note: Do not chill the ethanol precipitation prior to centrifugation. Incubation at low
emperatures does not improve the yield of ethanol precipitation with ammonium
acetate and may precipitate impurities that will inhibit subsequent steps.
19. Remove the supernatant carefully.
20. Gently overlay the pellet with 300 ul of 80% ethanoI.
2l. Spin in a microcentrifuge at 14,000 rpm for 10 min.
22. CarefuHy remove the supernatant.
to be sure that you did not lose the sample during washing.)
23. Air dry the pellet for approximately 10 min to evaporate residual ethanol.
24. Dissolve the precipitate in 10 ul of H20 and store at ―20 oC.
Adaptor ligation
The procedure below describes a 10-ul reaction in which the Marathon cDNA adaptor is ligated to the ds cDNA obtained after second-strand synthesis. Note that this reaction uses only half of the ds cDNA produced above. The kit contains enough components for 10 separate ligations of the Marathon cDNA Adaptor to ds cDNA.
We recommend that you perform a positive control adaptor ligation using the ds cDNA madefromthe human placental polyA+ RNA provided with the kit. This is necessary if you want to have a positive control for later steps.
Note: Allow 5 x ligation buffer to completely thaw at room temperature and keep it at room temperature for 30 min before use. Do not put the 5 x ligation buffer on ice.
1. Combine the following reagents in a 0.5-ml microcentrifuge test tube at room temperature and in the order shown:
5 ul ds cDNA
2 ul Marathon cDNA Adaptor (10uM)
2 ul 5 x DNA ligation buffer
l ul T4 DNA ligase (1 unit/ul)
10ul Total volume
2. Mix by vortexing and spin briefly in a microcentrifuge.3. Incubate at either: - 16 oC overnight; or - room temperature (19-23 oC) for 3-4 hr.
4. Heat at 70 oC for 5 min to inactivate the ligase.
5. Using the following guidelines, dilute your adaptor-ligated ds cDNA to a concentration which is suitable for subsequent RACE PCR procedures (~ 0.l ug/ml).
* lf you compared your yield of ds cDNA to that obtained with the positive control RNA:
- If the yield of your experimental sample is equal to or greater than the positive control, dilute l ul of the reaction mixture with 250 ul of Tricine-EDTA buffer.
- lf the yield of your experimental sample is less than the positive control, dilute the reaction mixture with proportionately less Tricine-EDTA buffer. e.g., if your sample contained 5-fold less cDNA than the positive control, dilute l ul in 50 ul of Tricine-EDTA buffer.
(lf you cannot see your ds cDNA with ethidium staining, you will probably need to repeat sect1'OnS D-F using fresh (or more) RNA.)
* If you did not compare your yield of ds cDNA to that obtained with the positive control RNA, prepare separate l/50 and 1/250 dilutions of adaptor-ligated ds-cDNA in Tricine-EDTA buffer. Perform the subsequent RACE PCR reactions using the specified amount of both dilutions until you determine which dilution gives you the best results.
6. Dilute 1 ul of the positive control reaction mixture with 250 ul of Tricine-EDTA buffer.
7. Store the undiluted adaptor-ligated cDNA at ―20 oC for future use.
(If your RACE reactions generate smears, you may wish to prepare more dilute sample of the adaptor-ligated ds cDNA.)
8. Heat the diluted ds cDNA at 94 oC for2 min to denature the ds cDNA.
9. Cool the tube on ice for 2 min.
10. Briefly spin the tube in a microcentrifuge to collect the contents in the bottom of the tube.
Store at-20 oC until ready for RACE PCR.
At this stage you essentially have a library of adaptor-ligated ds cDNA. The RACE reactions use only a fraction of this material for each RNA of interest. There is enough material to perform the rest of the protocol using gene-specific primers for several different RNAs. The kit contains enough primers and dNTPs to perform 100 standard 50-ul PCR reactions.
PCR Reaction (5'- or 3’- RACE).
Marathon PCR reactions have been optimized with CLONTECHIs Advantage KIenTaq Po[ymerase Mix, which includes TaqStart Antibodyfor automatic hot start PCR. You must use some form of hot start PCR (i.e., Taq Start Antibody, wax beads, or manual hot start) to minimize background in your RACE reactions.
** Design PACE primers so that the tm is above 70 oC (usually 35-40 mer with 50 % GC)
l. Prepare enough PCR master mix for all of the PCR reactions plus one additional tube. The same master mix can be used for both 5’- and 3'-RACE reactions. For each 50-ul reaction; mix the following reagents:
36ul H20
5 ul 10XKlenTaq PCR reactionbuffer
l ul dNTP (10mM)
l ul Advantage KlenTaq Polymerase Mix (50 x)
43 ul Final volume
Mix weIl by ortexing (without introducing bubbles) and briefly spin the tube in a microcentrifuge.2. Prepare 5-RACE PCR reactions. Add the components in the order shown in 0.2-ml PCR tubes,
4. Commence thermal cycling in a MJ -Res. DNA Thermal Cyc[er PT-100 using the following program (programs 1 and 2 work with the positive control 5'-RACE TFR and APl primers):
(If gene-specific primer has a Tm greater than 70 oC):
･ 94 oC for l min
･ 5 cycles:
94 oC 5 sec
72 oC 4 min･ 5 cycles:
94 oC 5 sec
70 oC 4 min･ 25 cycles:
94 oC 5 sec
68 oC 4 min
(lf gene-specific primer has an annealing temperature of 65-70 oC):･ 94 oC for l min
･ 30 cycles
94 oC 30 sec
68 oC 4 min
(If gene-specific primer has annealing temperature of an 60-65oC):･ 94 oC for l min
･ 30 cycles:
94 oC 30 sec
60 oC 30 sec
68 oC 4 minNotes on cycling:
* You may need to determine the optimal cycling parameters for your gene empirically. If you see weak bands or no bands, perform five additional cycles at 68oC.
* The optimal extension time depends on the length of the fragment being amplified.
We typically use 4 min for cDNA fragments of 2-5-kb. For 0.2-2-kb targets, we reduce the extension time to 2-3 min. For 5-10-kb targets, we increase the extension time up to 10 min.
5. When cycling is completed, analyze 5 ul from each tube, along with appropriate DNA size markers, on a 1.2% agarose gel.
6. [OptionaI] If the primary PCR reaction fails to give the distinct band(s) of interest, you may wish to perform a secondary or "nested" PCR reaction using the AP2 primer supplied with Marathon-Ready cDNA and a nested gene-specific primer.
a. Dilute 5 ul of he primary PCR product into 245 ul of Tricine-EDTA buffer.
b. Repeat steps l-5 above, using:
- 5ul of the dilued primary PCR product in place of the Marathon-Ready cDNA.
- l ul ofthe AP2 primer and l ul of your nested anti-sense gene-specific primer.
- Fewer cycles (15-20 instead of 25-30).