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 transcription tube and mix well.

Transfer contents of in vitro transcription mix to 1.5mL RNase/DNase-free tube.

Add 330 uL of RLT mix. (stopping point �80 overnight)

Add 250ul 95% Ethanol and mix well by pipetting. (Do not spin here!)

Apply sample (700ul) to RNeasy mini spin column sitting in a collection tube.

Centrifuge 15 sec at >= 8000 x g. Discard flow through.

Protocol for mRNA amplification

aRNA purification using Qiagen RNeasy COLUMNS ( cont’d)

Transfer RNeasy column to a new 2-ml collection tube (supplied).

Add 500ul Buffer RPE (which must contain ethanol) and centrifuge 15 sec at >=8000 x g.

Discard flow-through but re-use tube.

Add another 500ul Buffer RPE to the RNeasy column.

Centrifuge for 2 min at maximum speed.

Discard flow-through but re-use tube

Add another 500ul Buffer RPE to the RNeasy column.

Centrifuge for 2 min at maximum speed.

Place RNeasy spin column into a new 1.5-ml collection tube (supplied).

Centrifuge for 1 min at maximum speed to completely dry column.

Transfer RNeasy column into a new RNase, DNase-free 1.5-ml collection tube (not supplied).

Add 30ul RNase-free water directly onto membrane.

Centrifuge for 1 min at >=8000 x g to elute.

Repeat if expected yield is >= 30ug.

Check RNA concentration and quality by measuring OD260 and OD260/280.

Check RNA quality by running a gel.

Add 1uL of Sample to 1-2uL of loading dye.

Incubate at 65C for 10min. Put on ice immediately after incubation.

Run on a 1.25% agarose gel (2.5g agarose in 200mL 1X MOPS) in 1X MOPS Buffer for 45 minutes to 1.5hr at 165 V.

second round amplification

In PCR reaction tube, mix

Ammt	Reagent
0.5-1ug	aRNA from 1st round
9ul	DEPC H2O
1ul	(2ug/ul) random hexamer (i.e. dN6)

70C for 3min, cool to room temperature.

Add to PCR Tube (separately or in a Master Mix)

Ammt	Reagent
4ul	5X First strand buffer
1ul	(1ug/ul) oligo dT-T7 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 )

42C for 90min in thermal cycler.

From here, follow the procedure of first round amplification for

Second strand synthesis,

DS cDNA cleanup,

In Vitro TRanscription,

and aRNA purification using RNEasy .

Note: when labeling amplified RNA - USE RANDOM HEXAMERS.

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