This protocol will allow you to isolate enough total RNA from rat cells to label for our microarray experiments.  Each group will be harvesting RNA from one to two tissues. There are four basic steps we will go through--1.  Breaking Open the Animal Cells, 2. Preparing the RNA, 3. Determining the concentration of RNA, 4. Checking the RNA for degradation using gel electrophoresis.

Remember as you step through this procedure that there are RNases (Enzymes that break down RNA) everywhere.  Your hands, hair, and breath are two major sources. You should wear gloves throughout these procedures and avoid using your gloves to touch your skin or hair during the procedures.  Do not directly breathe or talk over the tubes. Do not have anything unneeded on your benches and think the protocol through before you begin.  If you have long hair make sure it is tied back for this lab.

Your benches and pippettes should be washed down with a special detergent called RNAZap (Ambion) that will help get rid of Rnases before you start the protocol. Note the timing on this experiment is very tight - you need to be familiar with every word of this protocol before you come into lab!

Breaking Open the Animal Cells (30 min.) *** The goal of these steps is to break open the cells as quickly as possible by dissolving them in a Tri-Reagent. CAUTION: THE TRI REAGENT IS ACIDIC AND WILL PUT HOLES IN YOUR CLOTHES IF IT GETS ON THEM. The reagent not only helps us to break open the cells with acidic and hydrophobic ingredients but also contains a chemical called Guanadine Thiocynate that stops RNases from working temporarily. Overall, the faster you can get your tissue dissolved in this buffer , once you take the tissue out of the freezer, the less degraded your RNA will be. Warm peices of tissue that are not broken down by the Tri-Reagent within a minute or two of homogenization are likely to contain active RNases that are not stopped by the TRi-reagent.

1.    Setting- up your lab bench: In preparation for these critical steps, clear off your bench space as much as possible, please put on gloves and your safety glasses and wipe down your bench, gloves and pippete handles and the outside of other inert objects you might be touching like pens using RNAzap wipes. From now on you want to try not to touch anything that is not RNase Free with your gloves, If you do, please wipe them again with a wipe or change your gloves completely. Obtain an ice bucket filled with ice, a tube with Tri-Reagent from the refrigerator, a baked foil wrapped package containing a Dounce Tissue Homogenizer and Dounce pestles (RNases are removed from glass by baking at 350 degrees for 4 hours and the foil keeps us from reintroducing them), blue and yellow Rnase Free tips, RNase Free forcips (also baked), your pippetes, Rnase free 1.5 ml microcentrifuge tube and an RNase free 15 ml tube. Carefully open the foil packs containing the dounce homogenizer but leave the homogenizer and pestles setting on the foil . Make note of the labels on the pestles, A and B. A is a smaller pestle and you will use it to start homogenzing, The pestle marked B is slightly larger and will fit tighter in the homgenizer so it will be used second to make sure the tissue is completely homogenized. Also make sure you have your pippette ready and set to measure out Tri Reagent in 1 ml volumes. Weigh the empty 1.5 ml microcentrifuge tube on the scale---

Weight of centrifuge tube =___________________________

2.    Obtain a tube of rat tissue from the instructor when the scale is free. Weigh the tube and tissue and place it in your ice bath while you quickly figure out how much tissue you have and how much Tri-Regent to use.

Weight of tissue and centrifuge tube = ________________________

Weight of Tissue = Weight of tissue and centrifuge tube - weight of centrifuge tube = _________________________________

Use 1 ml of Tri-Reagent for every100 mg or 0.1 g of tissue= ____________________________ml of Tri-Reagent

3.    Continue to move quickly and into your dounce tissue homogenizer add:

a.    The appropriate amount of Tri Reagent calculated above or use 9 ml.

b.    Your tissue- dump it in as quickly as you can into the TRi-Reagent by turning the eppindorf tube over and tapping it, if needed use the RNases free forcips to help move the tissue and then begin to homogenize the solution with the pestle A up and down in the solution as quickly as you can without breaking through the liquid surfase with the homogenzier.

c.    Once the tissue has almost totally disappeared through homogenization using pestle A(about 20-30 strokes up and down) switch to homogenizer B and homogenize with this homogenizer until tissue is completely dissolved or at least 20 more strokes up and down again not breaking the surfase tension.

4.    Now dump the contents of the homogenizer into the RNAse free 15 ml tube and incubate at room temperature for five minutes. Make sure you label it with its contents. This solution if frozen at - 80 would be fine to freeze and keep for a number of months.

a.    If you are doing multiple samples, let the first sample sit at this step until both have been processed.

Extracting the RNA (2 hours and 15 min) - This step divides the more hydrophillic RNA from the more hydrophobic proteins and the bulkier DNA through centrifugation.

5.    Add 200 mL chloroform PER mL of Tri Reagent. CAP The TUBES TIGHTLY and shake vigorously to mix for 20 seconds. Then incubate at room temperature for 5 minutes.

a.    If you started with 9 mL Tri Reagent, add 1800 mL chloroform. Do this in the hood as chlorform is an anestetic.

Added_________________ ml chloroform

6.    Centrifuge at 12,000 x g for 15 minutes at 4°C. (CENTRIFUGE FROM CELL LAB or Dr. Monroe's Lab)

During this 15 minute time-make sure your gel that will be used to check the quality of the RNA is poured and tubes are labeled for the next step and that you have all ingrediants for step 8 and 10 available. You will need to assign a group member to each of these tasks- ie one person should make sure the samples are spinning while the others prepare for the next few steps, making sure tubes are labeled and ready for step 8, pippetes are set and ready for step 7, isopropanol, ethanol is ready., and two RNA gels ready to use for quality checking the RNA at the end of the class period.

7.    The centrifugation will divide the sample into three layers. Transfer as much as possible of teh upper aqueous phase into a fresh 15 ml tube without disrupting the lower layers.

a.    The aqueous phase will be the top, clear layer. It contains the RNA but nothing to inhibit RNases! So be careful not to talk or breath or lean over open tubes to introduces RNases into this mixture. It will also contain some hydrophillic proteins, salt and sugar.

b.    The middle layer will be white and contains unbroken tissue and DNA. Be careful not to get any of this layer in your solution.

c. The lowest layer will be pink and contains most of the the protein - particularlly we hope the RNases.

8.    Add 500 mL of isopropanol PER mL of Tri Reagent. Vortex to mix and incubate at room temperature for 5 minutes.

a.    If you started with 9 mL Tri Reagent, add 4.5 mL isopropanol.

b. The purpose of this step is to further purify your RNA from protein and sugar in the mixture. The RNA will precipitate out in solutions of the right concentrations (50% ethanol and 5 M NaCl) while the protein and sugar will hopefully stay dissolved.

Added__________________ml isopropanol

9.    Centrifuge at 12,000 x g for 8 minutes at 4°C. (CENTRIFUGE FROM CELL LAB/Monroe Lab). MAKE SURE YOU KNOW WHERE YOUR PELLET WILL BE WHEN YOU TAKE THE TUBE OUT OF THE CENTRIFUGE AS IT WILL BE FAIRLY SMALL. - Also, during this spin Remove the ingrediants for step 15, 16 from freezer and place on ice to dethaw!

10. Pour off the supernatant, being careful not to pour off the pellet. Add 1 mL 75% ethanol per mL of Tri Reagent (9 ml total).

a.    Pellet will be on bottom of the tube, but may also run up the side of the tube.

b.    70% or 80% ethanol is ok to use.

c. This step is used to remove the isopropanol and replace it with mainly ethanol which evaporates faster. It also serves to get rid of any remaining salt or sugar which should redissolve in teh ethanol while RNA will not.

11. Centrifuge at 7,500 x g for 5 minutes at 4°C. (CENTRIFUGE FROM Monroe/CELL LAB) and dump off the bulk of the alchol, pippette off any last remaining bits.

12. Centrifuge for 1 minute, simply to bring any remaining ethanol to the bottom of the tubes and pippette off any remaining alcohol.

13. Dry the samples in the hood by opening the tube up and placing it in the hood for two to five minutes (You need the ethanol to evaporate as it can inhibit the reaction where you make cDNA but if you dry the RNA too much, it will not redissolve). Recap the tubes.

14. Resuspend the samples in 50 uL of RNA Storage Solution and transfer into two 0.5 mL tubes (25 ul in each) - please hand one tube into the instructor and continue on with the other tube (this size tube is a must to help you remove the inactive reagen in step 16).

15. The next step is to remove the DNA. For this you will need to add 3 ul of DNA-free buffer (approx. 0.1 volumes) to your tube. Add 1 ul of DNAase and incubate at 37 degrees celcius for 15 min. (procedure recommends 30 min. so check your time if you have time leave for another 5/10 min.) During this 15 minute time-make sure your gel is poured and ready to run.

16. Stop reaction by resuspending in the DNase Inactivation reagent. This contains a bead that will bind up the DNAse and pull it out of solution. Add 3 mL to the sample, incubate at room temperature for 5 minutes, tapping to resuspend the activation reagent periodically- every minute or two.

17. Centrifuge at 10,000 x g for 1.5 minutes and then transfer the RNA to a fresh tube. Note to centrifuge, you will need to place the 0.5 ml eppindorf tube inside the bottom of a larger 1.5 ml tube in order to have it fit in the centrifuge. Just cut the top off the 1.5 ml tube you used in the second step. When trasferring the RNA to a new tube after the centrifugation, avoid pipetting the inactivation reagent.

Quantifying RNA (20 min)

20. Quantify the RNA using the Nano-Drop spectrophotometer (in Herrick’s lab).

The concentration of RNA is read by measuring the absorbance of a sample at A260 on a spectrophotometer.  The RNA sample is placed in a quartz cuvette or on the nanodrop in order to read the optical density (OD). Common glass and plastic will also absorb at this wavelength so special quartz cuvettes that do not absorb light at this wavelength are used to measure the absorbance of the RNA. Be very careful with these, as they are very expensive.

Although the machine will determine the concentration of your RNA- you will need to know how to do this for your lab exam. This comes from Beers law that you should be familiar with from chemistry.   Scientists have determined an absorbence coefficient to use to determine the concentration of RNA in your sample preparation.  An RNA with the concentration of 40 ug/ml has an optical density of 1 at A260 so the equation is:

What would your concentration be if you had an OD of 0.3 and a pathlenght of 1 and no dilution of the RNA before you put it on the nanodrop? __________________________

Scientists also measure the absorbence of their RNA sample at A280 as well as A260. Both proteins and nucleic acids absorb light at 280 nm. By obtaining the ratio of A260/A280 scientists can thus get an idea of the purity of their RNA sample- ie whether or not it has protein contamination. Why would you care about this? Because a protein we don't want to see in our sample is Rnase! For pure RNA without a lot of protein this ratio should be 1.9-2.2. If the ratio is lower what does this mean in terms of protein content?

Answer: ___________________________________________________
 

During Step 6 above you will want to set up your gel (we just need 2 per class). You will pour a 1.2% agarose gel and have that ready to go for the end of class. Evenutally you will run 2 ug of this RNA on this gel at 100 V for 1/2 hour.   1.2 % means that you will need 1.2 g of agarose/ 100 ml of 1X TAE. The gel box we will use is for mini-gels. We will need two per class. Each mini-gel requires 25 mls of gel mixture.

Before Class, please determine and check your numbers with group members:

1. One member of group 1 or 3 at the TABLE should make this up during step 6.

For 25 mls of gel mixture you will need _____________ g of agarose. Place in flask then add TAE and then water.

You will need ____________ml of 50X TAE in 25 ml to make 1X TAE (use pippette)

You will need ____________ml of deionized Water to make up the final volume (use graduated cylinder)

Cover with saran and microwave until clear. Let cool for 5 min and then pour into mold in gel box. Use the comb with 8 wells.

2. One member of group 2 or 4 the Table should make this up.

You will need to make 200 ml of 1X TAE in a graduated cylinder. Once the gel is cooled you will need to cover it with 1X TAE.

__________ml of 50X TAE will be needed to make 200 ml of 1X TAE (pippette TAE into graduated cylinder first and then fill with water to 200 ml)

When gel is cooled pour this over gel so it covers the gel by 1mm.

3. When you are ready to run the gel: You will need to use your concentration to figure out how much RNA to run - you will want to run 2 ug of RNA on your gel.

If your concentration was 500 ng/ul, how much RNA would you need to run on the gel to obtain 2 ug? ___________________.

To each sample of 2 ug RNA add 1 ul of Ethidium Bromide and the appropriate amount of 6X loading dye to make your sample 1X.

How much 6X loading dye would you need to add to add to 5ul of RNA and ethidium bromide to make the final solution 1X loading dye? __________________

4. Run the gel for 30 min. at 100 Volts and then take a picture.

Some Notes: Several groups can run on one gel as we only need two lanes. We will not run a molecular weight as there are three distinct bands in RNA we are looking for and we can tell what they are by their pattern on the gel. Total RNA is made up of 85% rRNA and 14% tRNA with the rest being mRNA. Because the ribosomal RNA makes up such a huge portion of the RNA, you should only see ribosomal RNA on your gel not mRNA or tRNA.   There are four types of rRNA you will see, a 28S band, an 18S band and a band for the 5.8 or 5S ribosomal RNA although sometimes such as in the picture below you don't see this one. They should look very distinct and not degraded for the best quality RNA.  See the picture below for an example using rat RNA. The 28S band contains approximately 4100 bp and the 18S band contains approximately 1900 bp. 4800/1900 = 2.5. The brightness of the EthBR because it fits between the base pairs reflecs the number of base pairs available for it to get in between. Thus, another thing you should observe about good quality RNA is that the 28S band is about 2.5 times as intense as the 18S band is. If you still have DNA left, DNA will show up on this gel. It will look larger than the 28S band. Sometimes tRNA also shows up- is the tRNA smaller or larger than the 5.8S and 5S ribosomal bands?. Take a look at the RNA quality guide produced by a group of undergrads at another University from some examples of what these types of things would look like and answer this question.

____________________________________

21. Store RNA at -80°C, avoiding repeated freeze-thaw cycles.

1 = Degraded RNA

2= Good RNA

3 = Good RNA