The protocol is a slightly modified version of that presented in Wang, E., Miller, L. D., Ohnmacht, G. A., Liu, E. T. & Marincola, F. M. High-fidelity mRNA amplification for gene profiling. Nat Biotechnol 18, 457-9 (2000).
A. First Strand Synthesis:
1. Add the following to steril-Rnase-Free 200 ul PCR tubes (Doc Frugals (22-161):
The “Eberwine” oligo-dT/T7 primer sequence is 5’-AAA CGA CGG CCA GTG AAT TGT AAT ACG ACT CAC TAT AGG CGC T(X15)-3’
The trehalose (sigma T5251) must be dissolved by heating and must be heated to 65-70C before using.
mRNA 3.5 ul
1.7M trehalose 3.5 ul
Eberwine Primer(1ug/ul) 3.0 ul
2. Heat the above mixture at 65C for 10 minutes. Snap cool on ice for ~1 minute. Spin down.
3. Add 10 ul of the following Rnase-Free cocktail:
5X First Strand Buffer (Gibco) 4 ul
0.1M DTT (Gibco) 2 ul
Rnasin (Promega) 1 ul
10 mM dNTP (Gibco) 1 ul
0.1 ug/ul Linear Acrylamide (Ambion-9520) 1 ul
SuperScript II(Gibco) 1 ul
4. Thermocycle the above mixture : 37C, 5min; 45C, 5 min; 10 cycles of 60C, 2 min; 55C, 2 min). Cool to 4C.
B. Second Strand Synthesis:
1. Add 130 ul of the following cocktail to the First Strand reaction above:
DEPC ddH2O 106 ul
10X Second Strand Buffer (see recipe below) 15 ul
10 mM dNTP mix (Gibco) 3 ul
E.coli DNA Ligase (10 U/ul NEB-205S) 1 ul
E.coli DNA Pol I-Holoenzyme (10U/ul NEB-209L) 4 ul
Rnase H (2 U/ul, Gibco) 1 ul
2. Incubate the above mixture at 16C for 3.5 hours. For mRNA amplification, stop the reaction by adding 10 ul of 0.5M EDTA and incubating at 65C for 10 minutes. If amplifying total, at 7.5 ul of 1 M NaOH/2mM EDTA and incubate at 65C for 10 minutes. Add this while tubes are at 16C in the PCR machine.
C. Cleaning up the Reaction:
1. Prepare the phase lock gel (PLG) tubes (Epindorff/5’-0032005-055-heavy tubes) by spinning down the goo.
2. Add 1 volume of Ph/Chl directly to the 200 ul PCR tube, mix thoroughly by pippetting up and down, and add to the numbered PLG tubes. Spin for ~ 5 minutes.
3. Transfer aqueous phase to Rnase-Free 1.5 ml tube.
4. Add 70 ul of 7.5M NH4Acetate to aqueous phase, mix. Add 1 ml of –20C 100% EtOH (Rnase free). Vortex.
5. Spin for 20 minutes at 4C. (A large white pellet should be seen at this point. If not, abandon prep.)
6. Wash with 100 ul 100% EtOH and spin for ~ 5minutes. Remove excess EtOH and dry briefly at RT.
7. Resuspend in 50 ul of DEPC DDH2O.
8. To further purify the DNA from Rnases and primers, used BIORAD columns to purify dscDNA (spin at 3800 rpm; 2 min to get rid of buffer, then 4 min to purify sample).
9. Dry sample by speedvac and resuspend in 16 ul DEPC water.
D. In Vitro Transcription (500 fold amplification):
1. Using the Ambion T7 Megascript kit (#1334), double the standard 20 ul reaction, so that the total volume in 40 ul.
2. Incubate the reaction for 14 hours at 37C, and follow the manufacturer’s instructions verbatim.
3. Add 1ml of TRIzol solution to each in vitro transcription tube and mix well. Add 200ul Chloroform per 1ml TRIzol solution. Mix by shaking vigorously for 15 second and incubate at room temperature for 2-3 min. Centrifuge at 12,000g for 15min at 4C.
4. Do one chloroform extraction.
5. Transfer aqueous phase to a new RNase free tube and add 600ul of isopropyl alcohol per 1ml TRIZOL reagent. Incubate sample at RT for 10min and then centrifuge at 14,000 rpm for 15min at 4C.
6. Wash pellet 2 times with 1ml 70% EtOH in DEPC-treated water.
7. Air dry pellet and quickly resuspend in 20 ul 10mM tris ph 7.5.
8. Check RNA concentration and quality by measuring OD260 and OD260/280. (Note: for OD readings, dilute in 200 ul (by mistake-should have been 100 ul) DEPC H2O with 50 mM NaOH to linearize RNA.)