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Protocol for mRNA amplification--RT-PCR实验过程
Isolate Total RNA using Qiagen midi kit (Cat#75142) ( see manufacturer's protocol) or by Trizol (Gibco BRL Cat# 15596-026) extraction (see manufacturer's protocol) or by PaxGene extraction (Pax tubes Cat# 762115, Pax Blood Extraction kit Cat# 762134) ( see manufacturer's protocol). Resuspend total RNA in DEPC water at 1ug/ul concentration.
Speedvac RNA down to a total of 3-4ug in 9uL DEPC H20. Prepare RNA in PCR tubes and store at �80C overnight.
First strand cDNA synthesis:
In PCR reaction tube, mix
| Ammt | Reagent |
| 0.5-3ug | total RNA |
| 9ul | DEPC H2O |
| 1ul | (1ug/ul) oligo dT(15)-T7 primer |
70C for 3min, snap cool on ice.
Add to PCR Tube (separately or in a Master Mix)
| Ammt | Reagent |
| 4ul | 5X First strand buffer |
| 1ul | 1ug/ul TS (template switch) primer |
| 2ul | 0.1M DTT |
| 1ul | RNaseIN (Promega Cat# N2111) |
| 2ul | 10mM dNTP (Pharmacia Cat# 27-2035-02 ) |
| 2ul | Superscript II (Gibco BRL Cat# 18064-071 ) |
Add 12uL to each tube. (Note: buffer and 0.1M DTT come with SS II)
42C for 90min in thermal cycler.
Second strand synthesis:
Add to PCR Tube
| Ammt | Reagent |
| 106 ul | DEPC H2O |
| 15ul | Advantage PCR buffer |
| 3ul | 10 mM dNTP mix |
| 1ul | RNase H (2U/ul Gibco BRL Cat# 18021-071 ) |
| 3ul | Advantage Polymerase (Clontech Cat# 8417-1 ) |
Add 128uL to each tube.
37C for 5min to digest mRNA, 94C for 2min to denature, 65C for 1min for specific priming and
75C for 30min for extension.
Stop reaction with 7.5ul 1M NaOH.
Incubate at 65C for 10min to inactivate enzyme.
DS cDNA cleanup:
Add to PCR Tube
| Ammt | Reagent |
| 1ul | 0.1ug/ul Linear Acrylamide (Ambion Cat# 9520) |
| 150ul | Phenol: Chloroform: Isoamyl alcohol 25:24:1 (Boehringer Mannheim Cat #101001) |
Mix well by pipetting (be careful not to spill or contaminate).
Spin the Phase lock gel tube down for 30 seconds at maximum speed.
Transfer the slurry solution to Phase lock gel tube (5’-3’ Inc. Cat# p1-257178)
Spin at 14,000rpm for 5min at room temperature.
Transfer the aqueous phase to RNase/DNase-free tube (stopping point).
Add 70ul of 7.5M ammonium acetate (Sigma Cat# A2706) and gently mix.
Add 1ml 95% room temperature ethanol.
Centrifuge at 14,000rpm for 20min at room temperature.
Wash pellet with 500ul 95% ethanol and spin pellet down at maximum speed for 6min.
Air dry pellet and resuspend ds cDNA in 8ul DEPC H2O (stopping point).
In Vitro TRanscription
(Ambion; T7 Megascript Kit #1334)
In PCR reaction tube, mix
| Ammt | Reagent |
| 2 ul | of each 75mM NTP (A, G, C and UTP) |
| 2 ul | Reaction buffer |
| 2 ul | Enzyme mix (RNase inhibitor and T7 phage polymerase) |
| 8 ul | ds cDNA in H20 |
Add 20uL to each tube.
Incubate at 37C for 5-6hr.
aRNA purification using Qiagen RNeasy COLUMNS
Make up RLT Buffer Master Mix with b -ME and H2O.
| Ammt | Reagent |
| 3.5 ul | b -ME |
| 80 ul | H20 |
| 350 ul | RLT |
Add 100uL of RLT to in-vitro transcri
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