P-FPMI-43 - P-FPMI-43

Type
labeling
Description
In this protocol we use the Ambion kit # 1751, MessageAmp II which amplifies RNA producing aRNA ( cRNA). This aRNA is hybridized to the 21 K slides purchased from Jack Bell Research Centre.


Important Parameters for Successful Amplification


Input RNA quantity and IVT reaction incubation Time


Consider both the amount of sample RNA you have, and the amount of aRNA needed for your experiments when planning MessageAmp II experiments. These factors will influence how much input RNA is used, whether one or two rounds of amplification should be done, and how long to incubate the IVT reaction.


Accurate quantification


For experiments where the aRNA yield from different samples will be compared, it is

Essential to accurately quantify the input RNA used in the MessageAmp II procedure.


Recommended minimum and maximum amounts of input RNA


Table 1 shows the mass of total RNA that can be used in the MessageAmp II aRNA procedure. Alternatively 10–100 ng of poly(A) selected RNA can be used in the procedure. RNA must be in a maximum volume of 11 µl.


Table 1: Amount of Total RNA to use in MessageAmp II.


Amplification Recommended Minimum Maximum

Single Round 1000 ng 100 ng 5000 ng

Two Rounds 100 ng 0.1 ng 100 ng


RNA amount and IVT reaction incubation time


The MessageAmp II procedure can accommodate a wide range of input RNA amounts, but for reproducible and comparable results, use a fixed amount of RNA for all experiments. Tailor both the amount of input RNA used and the amplification procedure to produce the amount of aRNA needed for your microarray hybridizations. For instance, Affymetrix GeneChips require 10–15 µg of aRNA for each hybridization, but other commercial and core facility arrays may require slightly more or less aRNA.

Figure 3 shows aRNA yields from different amounts of Control RNA amplified with increasing IVT incubation times. When amplifying small RNA samples however, (e.g. ~250 ng or less) incubating the IVT reaction for 14 hr will maximize the amount of aRNA produced.

Typically 100 ng of total RNA input is the lower limit for synthesizing ~10 µg of aRNA using a 4 hr IVT reaction. If your total RNA samples will be ~100 ng or less, we advise conducting a preliminary amplification from a representative sample to determine empirically how much aRNA you can expect from your experimental samples. If the preliminary experiment does not produce enough aRNA for two array hybridizations, repeat the experiment using an IVT incubation as long as 14 hr, and/or consider using two rounds of amplification.




RNA Purity


The single most important factor affecting how efficiently an RNA sample will be amplified using the MessageAmp II aRNA Kit is the quality of the RNA. RNA samples should be free of contaminating proteins, DNA, and other cellular material as well as phenol, ethanol, and salts associated with RNA isolation procedures.

Impurities can lower the efficiency of reverse transcription and subsequently reduce the level of amplification. An effective measure of RNA purity is the ratio of absorbance readings at 260 and 280 nm. The ratio of A260 to A280 values should fall in the range of 1.7–2.1. RNA must be suspended in high quality water or TE (10 mM Tris-HCl, 1mM EDTA).


RNA integrity

The integrity of the RNA sample, or the proportion that is full-length, is another important component of RNA quality. Reverse transcribing partially degraded mRNAs will typically generate relatively short cDNAs that lack the sequence encoded upstream of the break in the RNA molecule.

There are currently no quantitative methods for measuring what percentage of mRNAs in a sample are full-length, however several procedures do exist for establishing the relative integrity of a sample. A method that requires only nanogram quantities of RNA is microfluidic sample analysis using the Agilent 2100 bioanalyzer and an RNA LabChip®

Kit. The bioanalyzer fractionates RNA molecules according to size, so that the amounts

of 18S and 28S ribosomal RNA (rRNA) can be determined. Primarily full-length RNA will exhibit a ratio of 28S to 18S rRNA bands that approaches 2:1.

Denaturing agarose gel electrophoresis and nucleic acid staining can also be used to separate and visualize the major rRNA species. When the RNA resolves into discrete rRNA bands (i.e. no significant smearing below each band), with the 28S rRNA band appearing approximately twice as intense as the 18S rRNA band, then the mRNA in the sample is likely to be mostly full-length. The primary drawback to gel electrophoresis is that microgram amounts of RNA must be sacrificed.




Reaction Incubation times should be precise and consistent


The incubation times in the protocol were optimized in conjunction with the kit reagents to ensure the maximum yield of nucleic acid product in each step—adhere to them closely.

A range of 4–14 hr is given for the IVT reaction incubation time (step II.F.3 on page 13). Refer to the graph in Figure 3 on page 7 to help determine what incubation time to use. Keep this IVT incubation time uniform if aRNA yield from different samples will be compared, or if you want to have equal amplification of different samples. Although differences in IVT incubation time among samples has had very little, if any, effect on array results in our hands, we still recommend using a uniform IVT incubation time for the most reproducible amplification and array analysis.


Master Mixes


We strongly recommend preparing master mixes for the MessageAmp II procedure. This approach reduces the effects of pipetting error, and improves reproducibility. Using Master Mixes is especially important when aRNA yield from different samples will be compared. Ambion provides a web-based Master Mix calculator at the following address:


www.ambion.com/techlib/append/msgamp2_mm_calc.php



Thorough mixing is very important for reproducibility


Below are specific instructions for mixing kit reagents, Master Mixes, and individual reactions. For maximum reproducibility and aRNA yield, follow these instructions closely.

• Mix each kit component after thawing.


Mix enzyme solutions by gently flicking the tube a few times before adding them to reactions. Thaw frozen reagents completely at room temperature (i.e. primers, nucleotides and 10 X buffers), then mix thoroughly by vortexing, and keep on ice before use.

• Mix Master Mixes by gentle vortexing.


After assembling Master Mixes, gently vortex to make a homogenous mixture without inactivating the enzyme(s).


• Mix individual reactions by pipetting up and down 2–3 times, then flicking the tube 3–4 times, and finally spinning briefly.

After adding Master Mixes or other reagents to individual reactions, pipet up and down 2–3 times to rinse reagents from the pipet tip. Then flick the tube with your finger 3–4 times to mix thoroughly, and finish by centrifuging briefly to collect the reaction at the bottom of the tube.


Incubator recommendations


• We recommend a calibrated hybridization oven or incubator (at constant temperature) for most of the MessageAmp II incubations.

To avoid any potential influence on the reaction temperature from the tube holder, let tube holders equilibrate in the incubator for sufficient time, or use a tube holder that doesn’t touch the sides and bottoms of the tubes—for example a floating tube support.


• For the 16°C second strand synthesis reaction incubation (step II.D.2

on page 11), we recommend using a thermal cycler

. Ideally these reactions should be incubated in a calibrated thermal cycler with a lid temperature that matches the block temperature. Most machines do not have this feature, so if the lid temperature is static (~100°C), use it with the heat turned off, or do not use the heated lid at all. Otherwise heat from the lid will raise the temperature of the solution in the tube, compromising the reaction.

• We do not recommend using heat blocks or water baths for MessageAmp II reaction incubations.

The MessageAmp II procedure is very sensitive to temperature; therefore use incubators that have been professionally calibrated according the manufacturer’s recommended schedule. Variable or inaccurate incubation temperatures can limit aRNA synthesis. Preheat incubators if necessary so that the correct temperature has stabilized before reactions are placed in the incubator. It is also very important that condensation does

not form in the reaction tubes during any of the incubations. Condensation changes the composition of reaction mixtures, and we have seen that it can greatly reduce yield.


Maintaining consistency


Procedural consistency is very important for planning amplification experiments. Consider implementing a detailed procedural plan that will be used by everyone in the lab to maintain consistency. This type of plan will minimize variation due to subtle procedural differences that can influence RNA amplification, and which may

complicate gene expression studies. The plan should include basic information such as the method of RNA isolation, the amount of RNA to use in the procedure, and how long to incubate the IVT reaction. It should also address specifics that are not often included in protocols such as which tubes, tube racks, and incubators to use for each step in the process. Finally, develop a consistent work flow, for example standardize stopping points in the method. The idea is to standardize all of the variables discussed in this section of the Instruction Manual and carefully follow all the protocol steps in order to maximize amplification consistency among samples.


Prepare the Wash Buffer


Add 24 ml ACS grade 100% ethanol (ACS grade or better) to the bottle labeled aRNA Wash Buffer. Mix well and mark the label to indicate that the ethanol was added.


Reverse Transcription to Synthesize First Strand cDNA


Incubators needed:

• 70°C (thermal cycler recommended)

• 42°C (hybridization oven or incubator recommended)


1. Mix RNA, 1 µl of T7 Oligo(dT) Primer


We sue 5 µg of total RNA in a total volume of 11 µL.


According to the each sample concentration measure the amount of RNA and water required.


a. Place a maximum volume of 11 µl of total RNA into a nonstick, sterile, RNase-free. 1.5 ml microfuge tube. RNA must be in high quality water or TE. (See Table 1 on page 6 for minimum and maximum RNA input amounts.)

b. Add 1 µl of T7 Oligo(dT) Primer.

c. Vortex briefly to mix, then centrifuge to collect the mixture at the bottom of the tube.

If your experiment will include RNA Spikes (from Ambion’s ArrayControl Kit), add them to samples at this step.


2. Incubate 10 min at 70°C, then place on ice


a. Incubate 10 min at 70°C in a thermal cycler.

b. Centrifuge samples briefly (~5 sec) to collect them at the bottom of the tube. Place the mixture on ice.


Add 8 µl of ReverseTranscription Master Mix and place at 42°C


At room temp, prepare Reverse Transcription Master Mix in a nuclease-free tube. Assemble enough to synthesize first strand cDNA from all the RNA samples in the experiment, including 5% overage to cover pipetting error. We provide a master mix calculator on our website to calculate reagent amounts:


www.ambion.com/techlib/append/msgamp2_mm_calc.php


Assemble the Reverse Transcription mater Mix in the order shown:


Table 2: Reverse Transcription Master Mix


Reagent Amount in µL

10X First Strand Mix 2

dNTP’s mix 4

RNase Inhibitor 1

Array Script 1

Total 8


b. Mix well by gently vortexing. Centrifuge briefly (~5 sec) to collect the Reverse Transcription Master Mix at the bottom of the tube and place on ice.


c. Transfer 8 µl of Reverse Transcription Master Mix to each RNA sample, mix thoroughly by pipetting up and down 2–3 times, then flicking the tube 3–4 times, follow up with a quick spin to collect the reaction in the bottom of the tube.


d. Place the tubes in a 42°C incubator.


4. Incubate for 2 hr at 42°C Incubate reactions for 2 hr at 42°C (hybridization oven or incubator is recommended). After the incubation, centrifuge briefly (~5 sec) to collect the reaction at the bottom of the tube. Place the tubes on ice and immediately proceed to second strand cDNA synthesis (below).


Second Strand cDNA Synthesis


Incubator needed:

16°C: thermal cycler recommended


1. Add 80 µl Second Strand Master Mix to each sample

a. On ice, prepare a Second Strand Master Mix in a nuclease-free tube in the order listed below. Assemble enough to synthesize second strand cDNA from all the samples in the experiment, including 5% overage to cover pipetting error. We provide a master mix calculator on our website to calculate reagent amounts:

www.ambion.com/techlib/append/msgamp2_mm_calc.php


Assemble the Second Strand Master Mix on ice in the order shown:



Table 3: Second Strand Master Mix


Component Amount in µL

Nuclease free water 63

10X second strand buffer 10

dNTP mix 4

DNA Polymerase 2

RNase H 1

Total 80


b. Mix well by gently vortexing. Centrifuge briefly (~5 sec) to collect the Second Strand Master Mix at the bottom of the tube and place on ice.

c. Transfer 80 µl of Second Strand Master Mix to each sample, mix thoroughly by pipetting up and down 2–3 times, then flicking the tube 3–4 times, and follow up with a quick spin to collect the reaction in the bottom of the tube.

d. Place the tubes in a 16°C thermal cycler. It is important to cool the thermal cycler block to 16°C before adding the reaction tubes because subjecting the reactions to temperatures >16°C will compromise aRNA yield.

2. Incubate 2 hr at 16°C

Incubate 2 hr in a 16°C thermal cycler. If the lid temperature cannot be adjusted to match the 16°C block temperature, cover the reactions with the heated lid turned off, or if the lid cannot be turned off—do not cover the tubes with it. (Do not use a water bath or a heat block in a 4°C refrigerator for this incubation because the temperature will fluctuate too much.)


3. Place reactions on ice briefly or freeze immediately


After the 2 hr incubation at 16°C, place the reactions on ice and proceed to. cDNA Purification (below), or immediately freeze reactions at –20°C. Do not leave the reactions on ice for long periods of time.

This is a potential overnight stopping point, but it is better to complete the cDNA purification (next section) before stopping.


cDNA Purification

cDNA Filter Cartridges should not be subjected to RCFs over 16,000 x g because it could cause mechanical damage and/or may deposit glass filter fiber in the eluate. All centrifugations in this purification procedure should be done at 10,000 x g (typically ~10,000 rpm) at room temp.


1. Preheat Nuclease-free Water to 50–55°C

Before beginning the cDNA purification, preheat the 10 ml bottle of Nuclease-free Water to 50–55°C for at least 10 min.

Preheat the Nuclease-free Water to a maximum of 55°C; temperatures above 58°C can partially denature the cDNA, compromising final yield of aRNA.


2. Add 250 µl cDNA Binding Buffer to each sample

Add 250 µl of cDNA Binding Buffer to each sample from step D.2 on page 11, and mix thoroughly by pipetting up and down 2–3 times, then flicking the tube 3–4 times. Follow up with a quick spin to collect the reaction in the bottom of the tube. Proceed quickly to the next step.


3. Pass the mixture through a cDNA Filter Cartridge

Before using them, check each cDNA Filter Cartridge to make sure that the filters are flush against the bottom of the mini-column; push them into place with the wide end of a pipet tip if necessary. Also check that the cDNA Filter Cartridge is firmly seated in its wash tube (supplied).

a. Pipet the cDNA sample\cDNA Binding Buffer (from step 2) onto the center of the cDNA Filter Cartridge.

b. Centrifuge for ~1 min at 10,000 x g, or until the mixture is through the filter.

c. Discard the flow-through and replace the cDNA Filter Cartridge in the wash tube.

Make sure that the ethanol has been added to the bottle of Wash Buffer before using it in the next step.


4. Wash with 500 µl Wash Buffer


Prepare the Wash Buffer

Add 24 ml ACS grade 100% ethanol (ACS grade or better) to the bottle labeled aRNA Wash Buffer. Mix well and mark the label to indicate that the ethanol was added.


a. Apply 500 µl cDNA Wash Buffer to each cDNA Filter Cartridge.

b. Centrifuge for ~1 min at 10,000 X g, or until all the cDNA Wash Buffer is through the filter.

c. Discard the flow-through and spin the cDNA Filter Cartridge for an additional minute to remove trace amounts of ethanol.

d. Transfer cDNA Filter Cartridge to a cDNA Elution Tube.


5. Elute cDNA with 2 X 9.0 µl 50–55°C Nuclease-free Water

It is important to use Nuclease-free Water that is at 50–55°C for the cDNA elution. Colder water will be less efficient at eluting the cDNA, and hotter water (≥ࣙ58°C) may result in reduced aRNA yield.


a. Apply 9.0 µl of Nuclease-free Water (preheated to 50–55°C) to the center of the filter in the cDNA Filter Cartridge.

b. Leave at room temperature for 2 min and then centrifuge for ~1.5 min at 10,000 x g, or until all the Nuclease-free Water is through the filter.

c. Elute with a second 9.0 µl of preheated Nuclease-free Water. The double-stranded cDNA will now be in the eluate (~14 µl).

d. Proceed directly to section F. In Vitro Transcription to Synthesize aRNA, or place the cDNA at –20°C.

The purified cDNA can be stored overnight at –20°C at this point if desired.


In Vitro Transcription to Synthesize aRNA Incubator needed:


• 37°C (hybridization oven or incubator recommended)


The IVT reaction can be set up to synthesize unmodified aRNA, or biotin-labeled nucleotides (or other modified nucleotides) can be incorporated into the aRNA during the IVT. Samples that will undergo two rounds of amplification cannot be labeled with biotin at this step because it would interfere with the second round of amplification.

For the highest aRNA yield, conduct the IVT in a 40 µl final reaction volume. We provide instructions for a 20 µl biotin-labeled reaction for users who want to reduce the reaction cost by using only half the biotin-labeled nucleotides ( here we do not provide the protocol for biotin labeled nucleotides).


Add IVT Master Mix to each sample

At room temp, prepare an IVT Master Mix by adding the following reagents to a nuclease-free microfuge tube in the order listed below. Assemble enough to synthesize aRNA from all the samples in the experiment, including ≤5% overage to cover pipetting error. We provide a master mix calculator on our website to calculate reagent amounts: www.ambion.com/techlib/append/msgamp2_mm_calc.php


If two rounds of amplification will be done, this first round transcription should be unmodified, containing only unmodified CTP and UTP.

Assemble the IVT Master Mix at room temp in the order shown:


Note: amino-allyl-UTP (aa-UTP) is purchased separately from Ambion (Cat. # 8437).



Table 4: IVT Master Mix


Component Amount in µL

Double stranded cDNA ( 14) from the previous step

T7 ATP solution 75 mM 4

T7 CTP solution 75 mM 4

T7 GTP solution 75 mM 4

T7 UTP solution 75 mM 3

Amino-allyl-UTP 50 mM 3 (purchased separately from Ambion)

T7 10X reaction buffer 4

T7 Enzyme mix 4

Total 26


b. Mix well by gently vortexing. Centrifuge briefly (~5 sec) to collect the IVT Master Mix at the bottom of the tube and place on ice.

c. Transfer IVT Master Mix to each sample following the volume guidelines below, mix thoroughly by pipetting up and down 2–3 times, then flicking the tube 3–4 times, and follow up with a quick spin to collect the reaction in the bottom of the tube.


Incubate for 4–14 hr at 37°C

The minimum recommended incubation time is 4 hr; the maximum is 14 hr. It is important to maintain a constant 37°C incubation temperature. We recommend incubating in an incubator such as a hybridization oven because it is extremely important that condensation does not form inside the tubes; this would change the reagent concentrations and reduce yield.


Note: we incubate for 4 hours at 37°


Add Nuclease-free Water to bring each sample to 100 µl

Stop the reaction by adding Nuclease-free Water to each aRNA sample to bring the final volume to 100 µl. Mix thoroughly by gentle vortexing.


Proceed to the aRNA purification step (below) or store at –20°C.

The aRNA can be stored overnight at –20°C at this point if desired.


aRNA Purification

This purification removes enzymes, salts and unincorporated nucleotides from the aRNA. At the end of the purification, the aRNA can be eluted from the filter with Nuclease-free Water.

aRNA Filter Cartridges should not be subjected to RCFs over 16,000 x g because it could cause mechanical damage and/or may deposit glass filter fiber in the eluate. All centrifugations in this section should be done at 10,000 x g typically ~10,000 rpm)



1. Preheat Nuclease-free Water to 50–60°C (≥10 min)

Before beginning the aRNA purification preheat the 10 ml bottle of Nuclease-free Water to 50–60°C for at least 10 min.


2. Assemble aRNA Filter Cartridges and tubes

For each sample, place an aRNA Filter Cartridge into an aRNA Collection Tube, and set aside for use in step 5.


3. Add 350 µl aRNA Binding Buffer

Check to make sure that each IVT reaction was brought to 100 µl with Nuclease-free Water.

Add 350 µl of aRNA Binding Buffer to each aRNA sample. Proceed to the next step immediately.


4. Add 250 µl 100% ethanol and pipet 3 times to mix

Add 250 µl of ACS grade 100% ethanol to each aRNA sample, and mix by pipetting the mixture up and down 3 times. Do NOT vortex to mix and do NOT centrifuge.

It is crucial to follow these mixing instructions exactly, and to proceed quickly to the next step.

Proceed immediately to the next step as soon as you have mixed the ethanol into each sample. Any delay in proceeding could result in loss of aRNA because once the ethanol is added, the aRNA will be in a semiprecipitated state.


5. Pass samples through an aRNA Filter Cartridge(s)

a. Pipet each sample mixture from step 4 onto the center of the filter in the aRNA Filter Cartridge.

b. Centrifuge for ~1 min at 10,000 X g. Continue until the mixture has passed through the filter.

c. Discard the flow-through and replace the aRNA Filter Cartridge back into the aRNA Collection Tube.


6. Wash with 650 µl Wash Buffer

a. Apply 650 µl Wash Buffer to each aRNA Filter Cartridge.

b. Centrifuge for ~1 min at 10,000 X g, or until all the wash solution is through the filter.

c. Discard the flow-through and spin the aRNA Filter Cartridge for an additional ~1 min to remove trace amounts of ethanol.

d. Transfer Filter Cartridge(s) to a fresh aRNA Collection Tube.


7. Elute aRNA with 100 µ preheated Nuclease-free Water

a. To the center of the filter, add 100 µl Nuclease-free Water that is preheated to 50–60°C.

b. Leave at room temperature for 2 min and then centrifuge for ~1.5 min at 10,000 X g, or until the solution is through the filter.

c. The aRNA will now be in the aRNA Collection Tube in ~100 µl of Nuclease-free Water.

The purified aRNA can be stored at –20°C overnight, or at –80°C for longer times if desired.


Amino Allyl aRNA:Dye Coupling and Clean-up

This kit was developed with Cy3 and Cy5 dyes from Amersham Pharmacia

Biotech, but mono-reactive NHS esters of any label moiety

should be capable of coupling to the amino allyl modified aRNA generated

with this kit. We use monofunctional Cy3 (PA23001) and Cy5 (PA25001) from Amersham Pharmacia Biotech.


Vacuum dry aRNA to 7 µl


Place amino allyl aRNA in a nuclease-free microfuge

tube, and vacuum dry to 7 µl or less. To couple an aRNA to more than one dye, transfer the aRNA per dye to a single tube and vacuum dry to 7 µl per

dye.

If the heater on the vacuum centrifuge is adjustable, use the medium or low temperature setting.

Check the progress of drying every 5–10 min, and remove the sample from the concentrator when it reaches the desired volume; do not overdry.


NHS Ester Dye Preparation

Prepare dye immediately before starting the dye coupling procedures. The preparation and storage of solubilized dye is important for the efficient labeling of amino allyl modified aRNA. It is imperative that the dye compounds remain

dry both before and after dissolving in DMSO because any water that is introduced will cause hydrolysis of the NHS esters, lowering the efficiency of coupling.


IMPORTANT

Store any unused solubilized dye in the dark at –80°C. Note that most dye

manufacturers do not recommend storing solubilized dyes for more than

~1 month.


Amersham Biosciences FluoroLink Cy5 (PA25001) and Cy3 (PA23001) monofunctional dye 5-pack


These dyes are supplied in relatively large aliquots; resuspend one dye vial in 32 µl of DMSO and keep in the dark at room temp for up to 1 hr until you are ready to use it. Molecular Probes


aRNA:Dye Coupling Reaction 1. Add 9 µl Coupling Buffer to the aRNA

To a tube containing 7 µl amino allyl aRNA (5–20 µg), add 9 µl Coupling Buffer and mix well.



2. Add 3 µl prepared dye to the aRNA and mix well

Add 3 µl of NHS ester dye in DMSO to the aRNA:Coupling Buffer

mixture. Mix well.


3. Incubate 30 min at room temp in the dark

This 30 min incubation at room temp allows the dye coupling reaction to occur. To keep the samples in the dark, typically it is sufficient to simply put the tubes in a closed drawer.


Add 4.5 µl 4M Hydroxylamine, mix, and incubate 15 min at room temp in the dark

To quench the reaction, add 4.5 µl 4M Hydroxylamine and mix well. Incubate the reaction at room temperature in the dark for 15 minutes. During this incubation, the large molar excess of hydroxylamine will quench the amine-reactive groups on the unreacted dye molecules.


Dye Labeled aRNA Purification


Repeat the aRNA purification protocol described above using the kit Mega Clear from Ambion (Cat. # 1908). The amplification kit comes only for 20 reactions and does not provide extra cartridges for dye-labeled aRNA purification.


NOTE: Do not use Nuclease-free Water to elute. Use the elution buffer provided in the Mega Clear kit.


This purification removes enzymes, salts and unincorporated nucleotides from the aRNA. At the end of the purification, the aRNA can be eluted from the filter with elution buffer.

aRNA Filter Cartridges should not be subjected to RCFs over 16,000 x g because it could cause mechanical damage and/or may deposit glass filter fiber in the eluate. All centrifugations in this section should be done at 10,000 x g typically ~10,000 rpm)


1. Preheat the elution buffer to 50–60°C (≥10 min)

Before beginning the aRNA purification preheat the bottle elution buffer to 50–60°C for at least 10 min.


2. Assemble aRNA Filter Cartridges and tubes

For each sample, place an aRNA Filter Cartridge into an aRNA Collection Tube, and set aside for use in step 5.


3. Add 350 µl aRNA Binding Buffer

Check to make sure that each IVT reaction was brought to 100 µl with Nuclease-free Water.

Add 350 µl of aRNA Binding Buffer to each aRNA sample. Proceed to the next step immediately.


4. Add 250 µl 100% ethanol and pipet 3 times to mix

Add 250 µl of ACS grade 100% ethanol to each aRNA sample, and mix by pipetting the mixture up and down 3 times. Do NOT vortex to mix and do NOT centrifuge.

It is crucial to follow these mixing instructions exactly, and to proceed quickly to the next step.

Proceed immediately to the next step as soon as you have mixed the ethanol into each sample. Any delay in proceeding could result in loss of aRNA because once the ethanol is added, the aRNA will be in a semiprecipitated state.


5. Pass samples through an aRNA Filter Cartridge(s)

a. Pipet each sample mixture from step 4 onto the center of the filter in the aRNA Filter Cartridge.

b. Centrifuge for ~1 min at 10,000 X g. Continue until the mixture has passed through the filter.

c. Discard the flow-through and replace the aRNA Filter Cartridge back into the aRNA Collection Tube.


6. Wash with 650 µl Wash Buffer

a. Apply 650 µl Wash Buffer to each aRNA Filter Cartridge.

b. Centrifuge for ~1 min at 10,000 X g, or until all the wash solution is through the filter.

c. Discard the flow-through and spin the aRNA Filter Cartridge for an additional ~1 min to remove trace amounts of ethanol.

d. Transfer Filter Cartridge(s) to a fresh aRNA Collection Tube.


7. Elute aRNA with 100 µL elution buffer

a. To the center of the filter, add 100 µl elution buffer that is preheated to 50–60°C.

b. Leave at room temperature for 2 min and then centrifuge for ~1.5 min at 10,000 X g, or until the solution is through the filter.

c. The dye-labeled aRNA will now be in the aRNA Collection Tube in ~100 µl of elution buffer.


Determining the incorporation of labeled nucleotides


a. Take the 100 l sample into a disposable NF-uvette, after reading the absorbance collect the sample back with the NF-barrier microtip for further use. Be careful NOT to contaminate the sample at this point!

b. Read the absorbance of all samples at 260 nm for quantifying RNA yield.

c. Read absorbance of the Cy3-labelled samples at 550 nm and Cy5-labelled samples at 650 nm.

d. Extinction coefficients are 150,000 for Cy3 and 250,000 for Cy5 respectively.

e. Abs (dye) / (extinction coefficient * 10^-6) = pmol dye/ l of sample.

f. Example : If A550 = 0.25

Then 0.25/0.15=1.67 pmol Cy3 dye / l.

g. Consider the nucleic acid to be single for calculating the yield of RNA. Therefore, factor of 40 is approximately correct for estimating nucleic acid concentration from A260 reading.

Parameters
Amount of nucleic acid labeled, Amplification, Used LAbel
Links
All experiments using protocol P-FPMI-43: (E-FPMI-13, E-FPMI-4, E-FPMI-5, E-FPMI-6, E-FPMI-7, E-FPMI-8, E-FPMI-9)