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Standard Operating Procedure

S.O.P#

IGF097.00

Title

RNA Amplification using the MessageAmp kit from Ambion.

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1.0	PURPOSE The MessageAmp aRNA Kit is based on the RNA amplification protocol developed in the laboratory of Dr James Eberwine. The procedure consists of reverse transcription with an oligo(dT) primer bearing a T7 promoter and in vitro transcription of the resulting DNA with T7 polymerase to generate hundreds to thousands of antisense RNA copies of each mRNA in a sample. The antisense RNA is referred to as aRNA and the amplification method is referred to as the aRNA amplification procedure
2.0	MATERIALS REQUIRED MessageAmp aRNA Kit (Ambion #1750) 5-(3-aminoallyl)-UTP, 50mM (Ambion#8437)* 100% ethanol Dyad PCR machine RNase free 0.2ml PCR tubes with caps RNase free 1.5ml screw capped eppendorf tubes * only required if incorporating amino allyl modified UTP into the RNA during the transcription reaction.
3.0	PREPARATION OF SYSTEM FOR USE Before using a kit for the first time ethanol should be added to both the cDNA wash and the aRNA wash as follows.
	3.1	To the cDNA wash add 11.2ml of ACS grade 100% ethanol, mix well. Indicate on the bottle that the ethanol has been added.
	3.2	To the bottle of aRNA wash add 20ml of ACS grade 100% ethanol, mix well. Indicate on the bottle that the ethanol has been added.

4.0	FIRST STRAND cDNA SYNTHESIS
	4.1	Program a Dyad PCR machine to keep at a constant 70ºC with the hot lid heating to 70ºC, save as 70CONST. Program the machine to keep at a constant 42ºC with the hot lid heating to 42ºC, save as 42CONST. Program the machine to keep at a constant 37ºC with the hot lid heating to 37ºC, save as 37CONST. Finally, program the machine to keep to a constant 16ºC with the hot lid off, save as 16CONST. Switch two PCR blocks on at 70CONST (volume 12ul) and 42CONST (volume 20ul).
	4.2	Place between 500ng of total RNA into a sterile, RNase free 0.2ml PCR tube. The volume of this sample must be 11ul or below. Where the RNA sample is limited the amount can be decreased to 100ng.
	4.3	To each PCR tube containing total RNA add 1ul Oligo(dT) Primer. Add nuclease free water to give a final volume of 12ul
	4.4	Incubate at 70C for 10 minutes.
	4.5	While the samples are incubating at 70ºC make up a Reverse Transcription MasterMix at room temperature: Per reaction:- 10x First Strand Buffer 2ul Ribonuclease Inhibitor 1ul dNTP mix 4ul Mix by pipetting gently up and down, centrifuge to collect the sample at the bottom of the tube, place on ice.
	4.6	Once the RNA samples have incubated at 70ºC for 10 minutes remove from the PCR machine, place on ice.
	4.7	Transfer 7ul of Reverse Transcription MasterMix to each RNA sample, mix by pipetting up and down. Place the tubes into the 42ºC PCR block.
	4.8	Working quickly add 1ul of Reverse Transcriptase to each PCR tube. Mix well by pipetting gently up and down and replace immediately into the 42ºC PCR block.
	4.9	Working quickly add 1ul of Reverse Transcriptase to each PCR tube. Mix well by pipetting gently up and down and replace immediately into the 42ºC PCR block.

5.0	SECOND STRAND cDNA SYNTHESIS
	5.1	Start a block on the PCR machine with the 16CONST program, 100ul volume.
	5.2	After the 2 hour incubation remove the samples from the PCR block and place on ice. Create a master mix of the second strand synthesis reagents (allowing extra for pipetting errors) and add 80ul of this mastermix to each sample. Per reaction:- [cDNA sample 20ul] Nuclease free water 63ul 10x second strand buffer 10ul dNTP mix 4ul DNA Polymerase 2ul RNase H 1ul Gently pipette the mix up and down to mix.
	5.3	Incubate at 16ºC for 2 hours in a PCR block.
	5.4	After the 2 hour incubation proceed immediately to the cDNA purification or freeze the reactions at -20ºC. Do not leave the reactions on ice for long periods of time.
6.0	cDNA PURIFICATION NOTE: The filter cartridges provided should not be subjected to rcfs of over 16000x g because it could cause mechanical damage and/or may deposit glass filter fibre in the eluate. All centrifugations in this section should be carried out at 10000 x g at room temperature.
	6.1	Before starting the cDNA purification preheat the bottle of nuclease free water, provided in the kit, for at least 10 minutes at 50°C.
	6.2	Immediately before starting the cDNA purification, equilibrate 1 Filter Cartridge per cDNA sample. Place a filter cartridge in a 2ml tube (supplied) and add 100ul of cDNA Binding Buffer.
	6.3	Incubate at room temperature for 5 minutes. During this time continue with 6.4.
	6.4	Transfer the cDNA reaction to a clean, sterile, 1.5ml RNase free tube. Add 250ul of cDNA Binding Buffer to each cDNA sample. Mix thoroughly by repeated pipetting.

	6.5	Pipette the cDNA sample/cDNA Binding Buffer from step 6.4 onto the centre of an equilibrated filter cartridge.
	6.6	Centrifuge for 1 minute at 10000x g or until the mixture has passed through the filter.
	6.7	Discard the flow through and replace the filter cartridge in the 2ml tube.
	6.8	Ensure that the ethanol has been added to the cDNA Wash Buffer. Apply 650ul of cDNA Wash Buffer to each filter cartridge.
	6.9	Centrifuge for 1 minute at 10000x g or until all the cDNA Wash Buffer is through the filter. Discard the flow through, replace the filter cartridge in the 2ml tube and centrifuge for a further minute at 10000 x g to remove trace amounts of ethanol.
	6.10	Transfer the filter cartridge to a cDNA elution tube. To the centre of the filter in the filter cartridge, apply 10ul of Nuclease-free water which has been preheated to 50ºC.
	6.11	Leave at room temperature for 2 minutes and then centrifuge for 1.5 minutes at 10000x g or until all the nuclease free water has passed through the filter cartridge.
	6.12	Repeat the elution with a second 10ul of preheated Nuclease-free water. Discard the filter cartridge.
	6.13	The volume of the eluted cDNA should be 16 – 18ul. Infrequently the volume will be less than 16ul and if this is the case add nuclease free water to give a final volume of 16ul and mix well.
	6.14	Place the samples on ice if proceeding immediately to the amplification reaction or store at -20°C until required. This is a good point to stop if carrying out the procedure over three days.
7.0	IN VITRO TRANSCRIPTION TO SYNTHESIZE aRNA
	7.1	Start a block on the PCR machine with the 37CONST program, 40ul volume.
	7.2	Transfer the 16ul cDNA solution to a 0.2ml sterile, RNase free PCR tube.

	7.3	Recipes for making either unmodified aRNA or aminoallyl aRNA are shown in the table below. Amino allyl aRNA - To the 16ul of double stranded cDNA from 6.14 above, add the components listed in the table in order, at room temperature (final volume = 41ul). Unmodified aRNA - To the 16ul of double stranded cDNA from 6.14 above, add the components listed in the table in order, at room temperature (final volume = 40ul).
		Amino allyl 16ul 3ul 4ul 4ul 4ul 2ul 4ul 4ul	Unmodified 16ul -- 4ul 4ul 4ul 4ul 4ul 4ul	Component double stranded cDNA 5-(3-aminoallyl)-UTP T7 ATP Solution (75mM) T7 CTP Solution (75mM) T7 GTP Solution (75mM) T7 UTP Solution (75mM) T7 10x Reaction Buffer T7 Enzyme Mix
		If a number of samples are being amplified create a mastermix, allowing extra for pipetting errors, gently pipetting up and down to mix. Centrifuge the tube briefly to collect the reaction at the bottom of the tube and add 80ul to each reaction tube, pipette up and down to mix.
	7.4	Incubate the reaction for 24 hours at 37ºC. The minimum recommended time is 6 hours. The maximum recommended time depends on the amount of input RNA used for the amplification.
	7.5	After the incubation time has elapsed add 2ul DNaseI to each reaction to remove the template DNA from the aRNA. Gently pipette up and down to mix.
	7.6	Incubate for 30 minutes at 37ºC. Proceed directly to the aRNA purification step or store at -20ºC.
8.0	aRNA PURIFICATION NOTE: The filter cartridges provided should not be subjected to rcfs of over 16000x g because it could cause mechanical damage and/or may deposit glass filter fibre in the eluate. All centrifugations in this section should be carried out at 10000 x g at room temperature.
	8.1	Preheat the Nuclease-free water to 50ºC for at least 10 minutes before starting the protocol.
	8.2	Immediately before starting the aRNA purification, equilibrate 1 Filter cartridge per aRNA sample. Place a filter cartridge in a 2ml tube and add 100ul of aRNA Binding Buffer. Incubate at room temperature for 5 minutes. Continue with 8.3 during this time.
	8.3	Add 60ul of elution solution to each sample and mix thoroughly by repeated pipetting. Transfer each sample to a fresh nuclease free 1.5ml microfuge tube.

	8.4	Add 350ul of aRNA Binding Buffer to each aRNA sample and mix thoroughly by repeated pipetting.
	8.5	Add 250ul of ACS grade 100% ethanol to each aRNA sample and mix thoroughly by repeated pipetting.
	8.6	Pipette each aRNA/Binding Buffer/ethanol mix onto the centre of an equilibrated Filter cartridge.
	8.7	Centrifuge for 1 minute at 10000x g or until the mixture has passed through the filter cartridge. Discard the flow through and replace the filter cartridge in the same 2ml tube.
	8.8	Ensure that the ethanol has been added to the aRNA Wash Buffer before use. Apply 650ul of aRNA Wash Buffer to each filter cartridge.
	8.9	Centrifuge for 1 minute at 10000x g or until the mixture has passed through the filter cartridge. Discard the flow through and centrifuge for an additional minute to remove trace amounts of ethanol.
	8.10	Transfer the Filter Cartridge to a fresh aRNA collection tube. To the centre of the filter, add 50ul of Nuclease-free water that has been preheated to 50ºC. Replace the Nuclease-free water in the 50ºC incubator.
	8.11	Leave the filter at room temperature for 2 minutes and then centrifuge for 1.5 minutes at 10000x g or until all the solution has come through the filter.
	8.12	Repeat the elution with a second 50ul of preheated Nuclease-free water. The aRNA will now be in the 2ml tube in 100ul of the solution used for elution. Discard the filter cartridge.
	8.13	Split the aRNA into 10ul aliquots and store at -80ºC.

9.0

ASSESSING aRNA YIELD AND QUALITY

9.1

To rapidly analyze aRNA synthesis, run 1ul of the purified aRNA per well on an Agilent Bioanalyzer RNA Nano Chip. See SOP IGF005.00 for further details on how to run the chips.
A typical electrophoretograph provided by Ambion is shown below, displaying the nucleotide size distribution resulting from one round of amplification. The average size (centre of the peak) should be approximately 1500nt.

electrophoretograp