Ambion method - mRNA Amplification

Kate 1.28.03

 

Before Using Kit for First Time:

Prepare cDNA wash buffer by adding 11.2 ml ACS grade 100% ethanol to the bottle.

Prepare aRNA Wash buffer by adding 20 ml of ACS grade 100% ethanol to bottle.

Mix all 4 IVT NTP’s together.

 

First strand cDNA synthesis:

In PCR reaction tube, mix:

Amount

Reagent

0.1 - 5ug

Total RNA

1uL

T7 Oligo (dT) primer

11uL

Nuclease free water

 

70oC for 10 min (Ambion 1) in a thermocycler with lid on, but not clamped down. 

Remove RNA samples from 70oC and centrifuge briefly.

Place mixture at 42oC while completing next step. (Ambion 2)

 

Prepare in a Master Mix and keep at room temperature:

Amount

Reagent

2uL

10X First Strand Buffer

1uL

Ribonuclease Inhibitor

4uL

dNTP Mix

 

Gently pipette or flick to mix, and then centrifuge briefly.

Incubate master mix at 42oC for 2 minutes prior to adding to reaction tubes.

 

Add to the master mix:

Amount

Reagent

1uL

Reverse Transcriptase

 

 

 

 

Quickly transfer 8ul of above Master Mix to each RNA sample, mix by pipetting. 

It is important to keep sample temperature as close to 42oC as possible.

42oC for 2 hours (Ambion 2) in thermocycler with lid on, but not clamped down. 

Centrifuge briefly, place on ice, and immediately proceed to next step of dsDNA synthesis.

 

Second strand synthesis:

Prepare the following master mix at room temperature:

Amount

Reagent

63uL

Nuclease free water

10uL

10X Second Strand Buffer

4uL

dNTP Mix

2uL

DNA Polymerase

1uL

RNase H

 

Add 80uL to each tube.

Gently pipette or flick to mix, and then centrifuge briefly.

16oC for 2 hours (Ambion 3) in thermocycler with lid completely off. 

After 2hr incubation, proceed to cDNA purification or (stopping point) freeze immediately at –20oC.

 

 

 

DS cDNA cleanup:

Preheat the 5mL bottle of water at 50oC for at least 10 minutes during the previous 2hr incubation. 

 

Equilibrate the columns during the previous 2hr incubation, immediately before starting the cDNA purification.

        Place filter cartridge in a cDNA Wash Tube (supplied) and add 50ul of cDNA Binding Buffer.

Incubate at room temperature for 5 min.

 

Transfer 100uL reaction from strip-8 PCR tubes into 1.5ml Eppendorf tubes, keeping on ice.

Make sure there are no crystals in the bottom of the cDNA binding buffer  - warm it up if there are crystals.

Add 250uL of cDNA binding buffer to each cDNA sample, and mix by pipetting.

Add the mix to the center of an equilibrated filter cartridge.

Centrifuge for 1 min at 10,000 x g.

Discard flow-through but re-use Wash Tube.

 

Add 500ul of cDNA wash buffer (be sure the ethanol has been added to bottle)

Centrifuge for 1 min. at 10,000 x g.

Discard flow-through but re-use Wash Tube.

Centrifuge again for 1 min at 10,000 x g to remove trace amounts of ethanol.

 

Transfer filter cartridge to cDNA Elution Tube (provided)

Add 10uL of preheated water to the center of the column. 

Incubate at room temperature for 2 minutes.

Centrifuge for 1 minute at 10,000 x g.

Repeat elution with a second 10ul of preheated water.

Centrifuge for 1 minute at 10,000 x g.

 

Check that the volume of the eluted cDNA sample is 16-18uL.  If it is less, add nuclease-free water to bring the sample volume up to 16uL.

 

(Stopping point)  If needed, stop and store the samples at –20oC.

 

In Vitro TRanscription

Prepare the following master mix:

Amount

Reagent

16uL

T7 NTP mix (75 mM each)

4uL

T7 10X Reaction Buffer

4uL

T7 Enzyme Mix

Add 24uL to each tube.

Gently pipette or flick to mix, and then centrifuge briefly.

37oC for 6 to 14 hours in a hybridization oven (be sure to use internal thermometer to verify temperature). 

 

Add to each reaction:

Amount

Reagent

2uL

DNaseI

 

 

 

Gently pipette or flick to mix, and then centrifuge briefly.

37oC for 30min in hybridization oven

 

Add 60uL Elution Solution to each aRNA sample to bring the final volume to 100uL.

Mix by pipetting or gentle vortexing.

 

                Proceed to aRNA purification step or (stopping point) store at –20oC.

 

 

 

aRNA PURIFICATION

 

Preheat 5mL bottle of nuclease free water at 50-60oC for at least 10 minutes.

 

Immediately before staring aRNA purification., equilibrate 1 filter cartridge per aRNA sample

        Place filter cartridge in a aRNA Collection Tube (supplied) and add 100ul of aRNA Binding Buffer.

Incubate at room temperature for 5 min.

 

Add 350uL of aRNA Binding Buffer to each aRNA sample.

Mix by pipetting or gentle vortexing.

Add 250uL of ACS grade 95% ethanol to each sample.

Mix by pipetting or gentle vortexing.

 

Apply entire sample to the equilibrated aRNA filter cartridge.

Centrifuge for 1 minute at 10,000 x g. 

Discard flow-through but re-use tube.

 

Add 650uL aRNA Wash Buffer to each filter. 

Centrifuge for 1 minute at 10,000 x g. 

Discard flow-through but re-use tube.

Centrifuge again for 1 min at 10,000 x g to remove trace amounts of ethanol.

Transfer cartridge to a fresh aRNA Collection Tube.

 

Elute aRNA by adding 50uL preheated (50oC) water to center of column.

Incubate at room temperature for 2 min.

Centrifuge for ~1.5 minute at 10,000 x g.

Repeat elution with a second 50uL of preheated water.

Centrifuge for ~1.5 minute at 10,000 x g.

 

Store aRNA at –80oC.

 

Gel/Spec

 

Dilute 1uL sample into 11uL DEPC H20.

Check RNA concentration and quality by measuring OD260 and OD260/280 using 12uL into 10uL cuvette.

 

Dilute 1uL sample into 2uL Sigma RNA Loading Buffer.

Check RNA quality by running a gel.

Incubate at 65C for 10min (amp4).  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.

 

 

 

(Optional) Second Round Amplification

 

This is done to generate additional aRNA if the first cycle of amplification fails to yield the amount of aRNA necessary for experiments.  This procedure if similar to the first round, but different primers are used and the reaction is set up slightly different.

1.                    Synthesis of First-Strand cDNA (second round) Make up the following reaction:

 

Amount

Reagent

Up to 2ug   (X)

aRNA

2 ul

Second Round Primers

10 ul - X

Nuclease free water (add to bring total volume up to 12ul)

Final volume = 12ul

Add reagents to 8 strip PCR tubes.  Incubate for 10 min at 70C in thermocycler without lid clamped down, but pulled over samples (or turn lid heat off) .  Remove RNA samples from 70C and centrifuge briefly. Place on ice briefly before next step.

 

2.                    At room temperature, make up the following master mix for reverse transcription and add 8ul to each reaction from above.

Amount

Reagent

2 ul

10X First Strand Buffer

1 ul

Ribonuclease Inhibitor

4 ul

dNTP Mix

1 ul

Reverse Transcriptase

8 ul total volume.  Mix with 12 ul reaction from above. Centrifuge to collect to bottom of tube.  Incubate reaction at 42C for 2 hours in thermocycler with lid on, but not clamped down.  After incubation, centrifuge briefly and proceed immediately to next step.

 

3.                    Add 1 ul of RNase H to each tube and incubate for 30 minutes at 37C. (this degrades aRNA leaving only cDNA as template for T7 Oligo (dT) Primer.  After incubation, proceed immediately to next step.

 

Synthesis of Second-Strand cDNA (Second Round)

 

1.                    To the reaction tube from above, add 5 ul T7 Oligo (dT) Primer.  Mix well by flicking or pipetting.  Centrifuge briefly to collect to bottom of tube.  Incubate at 70C for 10 minutes in thermocycler with lid on but not clamped down. Place on ice briefly before next step

2.                    Prepare the following master mix and add 74 ul to each tube from above so final volume is 100 ul.

 

Amount

Reagent

58 ul

Nuclease free water

10 ul

10X Second Strand Buffer

4 ul

5 mM dNTP Mix

2 ul

DNA Polymerase

(26 ul)

cDNA sample (from step above)

Total volume is 100 ul. Mix well by flicking and centrifuge briefly to collect in bottom of tube.  Incubate for 2 hours at 16C in thermocycler with lid completely off.

 

To complete aRNA production, now go back to cDNA purification step on page 2  and continue exactly from there on out, until you have completed with aRNA purification.