20.109(S10):Purify RNA and run affinity column (Day4)

Introduction

Today will be a busy day! Last time you prepared the 6-5 and 8-12 aptamers by an in vitro transcription reaction. Now you must purify the RNA before enriching the heme-binding aptamer on an affinity column.

To isolate RNA and change buffers, you will again do a column purification. However, this time the matrix will be a polyacrylamide gel rather than a silica resin. This size exclusion matrix retains nucleic acids longer than 20 bases. After purification, you will quantify your RNA by spectrophotometry. Nucleic acids (both RNA and DNA) have an absorbance peak at 260 nm. Beer's law may be used to quantify the amount of RNA from this peak: Abs = ε l c, where Abs is the measured absorbance, l is the path length (1 cm for most specs), c is concentration, and ε is the extinction coefficient. For RNA, ε is 0.025 (μg/mL cm)^-1, so 1 absorbance unit corresponds to 40 μg/mL of RNA. The absorbance at 280 nm gives some indication of RNA purity, as proteins have their absorbance peaks at that value (primarily due to the aromatic peptides tryptophan and tyrosine). An Abs₂₆₀:Abs₂₈₀ ratio of ~2:1 is desired. As a final preparation before column selection, the appropriate amount of RNA must be denatured at 70 °C and re-natured at room temperature in order to ensure that it has the right secondary structure. Otherwise the 8-12 aptamer might not bind to heme.

By now you should have signed up for your experimental conditions on today's Talk page. You and your partner will use the same ratio of aptamers, but a different number of column washes. (Vice versa?) The affinity column you will use consists of agarose beads with bound heme. Thus, when you add your aptamers to the column, 8-12 should bind but 6-5 should not. Of course, some 6-5 will non-specifically bind to the column. To remove it you will add aliquots of selection buffer to the column and let the 6-5 wash away. If you wash too vigorously, you may lose some 8-12 as well. Part of today's experiment is to hone in on the ideal wash conditions, that is, conditions that retain 8-12 well on the column relative to 6-5. Finally, to collect aptamer that remains bound to the column, you will add a concentrated heme solution. The free heme will compete with the bound heme for aptamer binding, and the aptamers should be readily eluted after a brief incubation step. One additional complication is that nucleic acids bind non-specifically to agarose. Thus, we will use tRNA in our buffer to compete with the aptamers for these non-specific sites. Overall, 8-12 aptamer should be enriched compared to 6-5 by the end of this process.

Once you have collected your samples, you will begin the process of concentrating the collected RNA. A common way to do this is by salt and ethanol precipitation. The ethanol lowers charge-charge screening, allowing the positive ion of the salt to ionically bind and precipitate out the negatively charged nucleic acids. After the column selection, your samples may contain little RNA, and should benefit from a long precipitation step (until our next session). You will also try to limit nucleic acid losses by adding a 'carrier' that co-precipitates with the RNA, namely glycogen. Ultimately, to see how the selected RNA performs in a binding assay you will first have to amplify it.

Protocols

Part 1: Prepare spin purification columns and purify RNA

1. Take your thawed IVTs from last time, and add 1/10 volume of DNase to each reaction. Place on the 37 °C heat block for 30 min, and meanwhile prepare the columns.
   • Immediately begin to prepare the spin columns - with all the incubations and spins, it will take longer than you think!
   • During spins and incubations, you may also be able to begin Part 2 (steps 1+2) below.
2. Take one Micro Bio-Spin Column and one 2 mL microfuge tube per reaction, and for each:
3. Rapidly flip the column back and forth a few tunes to resuspend the gel, then flick or tap it to rid air bubbles.
4. Snap off the plastic tab at the bottom of the column (a little buffer may flow out), and place the column in the 2 mL microfuge tube. Let the excess buffer drain off.
5. When the buffer stop flowing, centrifuge the tube for 2 min at 1000 g. (Is g the same as rcf or rpm? Ask if you’re not sure!)
6. Add 500 μL of Selection Buffer to the top of the column. Let it drain by gravity for a couple of minutes, then spin for 1 min at 1000g.
7. Repeat step 6 three more times. Meanwhile, label 1.5 mL eppendorf tubes for collection (on the side and with a sticky label on the cap), and cut off their caps.
8. Transfer each column to a 1.5 mL tube – both column and tube should be labeled, to be on the safe side – then add 75 μL of your IVT reaction gently to the center of the top surface of the gel.
9. Centrifuge for 4 min, at 1000 g. Carefully replace the cap on each sample, and store on ice for now.

Part 2: Quantify RNA recovery

1. For each RNA sample, you will add 5 μL of RNA to 695 μL of water. This ought to give you a measurement that's in a reliable range. You can prepare labeled eppendorf tubes containing the appropriate amount of water in advance.
2. To get the most accurate measurement, you should also prepare a blanking solution that contains everything except the RNA. Per sample, that means 695 μL of water and 5 μL of selection buffer.
3. As your RNA samples are ready, first measure the volume recovered to within a couple of μL (usually it is 85-90). Only then take 5 μL of the RNA and add to a water-filled eppendorf.
4. Add 695 μL of blanking solution to a cuvette, and hit Blank on the spectrophotometer. Then dump the blanking solution, and add 695 μL of your RNA solution instead. Record the 260 nm and 280 nm absorbance values in your notebook. You can simply touch your finger on the screen for coarse wavelength selection, then touch the arrows for fine selection.
5. Repeat for each sample.
6. An absorbance value of 1 indicates a concentration of 40 μg/mL of the measured RNA (i.e., the diluted solution and not the original stock).

Part 3: Prepare RNA for selection

1. Dilute each RNA sample in selection buffer to a concentration of 8 μM.
   • The respective molecular weights of the aptamers are 31,344 g/mol and 33, 824 g/mol for 6-5 and 8-12, respectively.
   • Most likely your final volume will be 0.5-1.5 mL; check with the teaching faculty if not.
2. Before continuing on, make sure you have enough of each aptamer to follow through on your experimental plan. If not, talk to the teaching faculty.
3. Prepare 2.5 nmol of your 6-5/8-12 aptamer mixture. Set aside 1.4 nmol (175 μL) in a well-labeled eppendorf tube - this will serve as your pre-selection sample in the binding assay on Day 7 and should be kept on ice.
4. The other ~ 1 nmol of mixture will be used for the column selection. It should be denatured at 70 °C for 5 min, then cooled at room temperature for at least 10 min.
5. As your benchmarks for the binding assay, you should also set aside exactly 1.4 nmol each of 6-5 and 8-12 alone. Give these two eppendorfs, along with the "pre" sample, to the teaching faculty, making sure they are well-labeled and tightly capped. These samples will be stored at -80 °C until needed.

Part 5: Begin RNA precipitation

1. Measure the approximate volume (to within a few μL) of solution recovered.
2. Add 1/10 volume of ammonium acetate, 1/200 V glycogen, and 2.5 V 100% ethanol, in that order.
   • For example, for a 200 μL solution, you would add 20 μL of the ammonium acetate.
3. Give the teaching faculty your samples to be put away at -20 °C until next time.