Protocol for mRNA amplification and target preparation

 

Based on Wang et. al., Nature Biotechnology, April, 2000

Last edited by Max Diehn, June, 2001

 

Isolate Total RNA using Qiagen mini kit (Cat#75142) (see manufacturer's protocol) or by Trizol (Gibco BRL Cat# 15596-026) extraction (see manufacturer's protocol). Resuspend total RNA in DEPC water at 1ug/ul concentration.

 

Primer sequences:

oligo dT(15)-T7 primer: (5 AAA CGA CGG CCA GTG AAT TGT AAT ACG ACT CAC TAT AGG CGC T(15) 3) [57-mer]

TS (template switch) oligo primer: (5 AAG CAG TGG TAA CAA CGC AGA GTA CGC GGG 3) [30-mer]

 

All incubations are done in a thermal cycler. For starting amounts of less than or equal to 1ug of total RNA a second round of amplification may be required to generate enough aRNA for a microarray hybridization (2.5-3ug aRNA/hybridization.)

 

Tube preparation

Prepare (and label) 8 sets of tubes:

       4 sets of 1.5 ml tubes:

         1 set for accepting the aqueous phase from the phase lock tubes following cDNA synthesis

         1 set for receiving the flow-through following the Bio-6 column purification

         1 set for pre-aliquoting the RNeasy reagent and receiving of the in vitro transcription products

         1 set for the final aRNA product eluted off RNeasy columns

       1 set of 0.5 ml tubes for pre-aliquoting the phenol:cholorform:isoamyl alcohol mix

       2 sets (plus 2 optional) of PCR tubes:

         1 set for the cDNA synthesis

         1 set for the in vitro transcription

       1 set of 0.5 ml eppendorf phase lock tubes. Be sure to spin the gels down first!

First strand cDNA synthesis:

In PCR reaction tube, mix

Ammt

Reagent

0.5-3ug

total RNA

9ul

DEPC H2O

1ul

0.5ug/ul oligo dT(15)-T7 primer

70C for 3-4min, snap cool on ice. (amp1)

 

Make 1st Strand Master Mix:

Ammt/rxn

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 total of 12ul to each tube.

42C for 90min in thermal cycler. (amp2)

(Note: buffer and 0.1M DTT come with SS II)


Second strand synthesis:

Make 2nd Strand Mater Mix:

Ammt/rxn

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 total of 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. (amp3)

 

Stop reaction with 7.5ul 1M NaOH solution containing 2mM EDTA.

Incubate at 65C for 10min to inactivate enzyme. (amp4)

 

 

DS cDNA cleanup:

Add to PCR Tube

Ammt

Reagent

1ul

0.1ug/ul or 1ug/ul Linear Acrylamide (Ambion Cat# 9520; comes at 5ug/ul)

150ul

Phenol: Chloroform: Isoamyl alcohol 25:24:1 (Boehringer Mannhem Cat #101001)

Mix well by pipeting (be careful not to spill or contaminate).

 

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.

 

Prepare Bio-6 Chromatograph column (Bio-Rad Cat# 732-6222)

Shake well before draining to get rid of air bubbles - otherwise it drains very slowly! When opening column, sometimes you will observe gel in the underside of the cap. This should be aspirated off to prevent contamination.

Wash column one time with 700ul DEPC H2O and spin at 700xg for 2min at room temperature.

Remove flow-through. Make sure all liquid is drained out of column. Spin again at 700xg for 2min to dry column completely.

 

Wash pellet with 500ul 95% EtOH and spin pellet down at maximum speed for 6min.

Air dry pellet and resuspend ds cDNA in 60ul DEPC H2O.

 

Load 60ul sample to the center of the Bio-6 column and spin at 700xg for 4min. (stopping point at 80 C)

Transfer samples to new PCR tubes.

Completely dry samples by speedvac. (stopping point at 80 C)


In Vitro TRanscription

(Ambion; T7 Megascript Kit #1334)

Make up master mix:

Ammt

Reagent

8 ul

of 75mM NTP Mix (A, G, C and UTP) (if new kit, combine NTPs into one tube)

2 ul

Reaction buffer

2 ul

Enzyme mix (RNase inhibitor and T7 phage polymerase)

8 ul

H2O

 

 

 

 

 

Add 20ul of mix to each PCR tube.

Incubate at 37C for 6hr. (amp5)

 

aRNA purification using Qiagen RNeasy COLUMNS

Make up RLT w/ b-ME and H2O Master Mix:

Per sample:

3.5ul b-ME

80 ul H2O

350ul RLT

 

Pre-aliquot 430ul RLT w/ b-ME and H2O to 1ml eppendorfs.

Transfer contents of in vitro transcription mix to the Rnase/Dnase-free tube.

Mix well. (stopping point 80 overnight)

 

Add 250ul ethanol (95%) 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.

 

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.

 

Pipet 500ul Buffer RPE onto RNeasy column and centrifuge for 2 min at maximum speed.

 

Remove flow through and pipet another 500ul Buffer RPE onto column. Centrifuge for 2 min at maximum speed. [This is an additional wash that is not in the Qiagen protocol which we have found necessary because of GITC contamination in the eluted RNA.]

 

Place RNeasy spin column into a new 1.5-ml or 2-ml collection tube (not supplied) and spin at full speed for 1 min to completely dry column.

 

Transfer RNeasy column into a new 1.5-ml collection tube (supplied) and 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.

 

Second round amplification

You only need to perform a second round of amplification if the starting input was too low to result in adequate yield of aRNA for labeling (2.5-3ug).

 

Mix aRNA(0.5-1ug) in 9ul DEPC H2O with 1ul (2ug/ul) random hexamer (i.e. dN6) and heat to 70C for 3min, cool to room temperature. Let sit for 10 minutes. Then add the following reagents:

 

4ul 5 X First strand buffer

1ul (0.5ug/ul) oligo dT-T7 primer

2ul 0.1M DTT

1ul RNAsin (Promega Cat# N2111)

2ul 10mM dNTP (Pharmacia Cat# 27-2035-02)

2ul Superscript II (SS II) (Gibco BRL Cat# 18064-071)

42C for 90min.

 

From here, follow the procedure of first round amplification for ds cDNA synthesis and cleanup.

Target labeling by reverse transcription

 

Follow standard microarray labeling techniques, using between 3-6ug of aRNA as input. Remember to use dN6 primer instead of oligo-dT. Use between 5-10ug of primer.