Midi-RNA Extraction from OCT Embedded Tissue and/or Snap Frozen Tissue

Collect and record Sample(s)
1. Fill a Little Playmate Cooler ¼ of the way with Dry Ice.
2. Keep all samples on dry-ice until needed, if more than ½ an hour is needed for set-up; place the samples in the -80 freezer in the Molecular laboratory.
3. Record each sample identification number as appropriate on a sample tracking/extraction worksheet. Assign each sample a different extraction number.

Set-up/Preparation:1. Label two (2) sterile, 15mL conical tubes for every sample to be extracted. Set the tubes up in order on two separate tube racks.
2. Remove the Glycogen from the -20 freezer and allow it to thaw completely on wet ice.
3. Turn on the Sorvall T21 centrifuge and the Sorvall Biofuge Pico. Allow both centrifuges to cool to 4 degrees Celsius before using.
4. If not already done, prepare a stock of 3M Sodium Acetate, pH 5.2.
5. If not already done, prepare a stock of 70% ACS Grade ethanol.
6. 64% Ethanol:  Add 64 ml of ACS Grade 100% (200 proof) ethanol to the bottle containing 36 ml RNase-free water in the RNAqueous kit. Mix well. Place a check in the empty box on the label to indicate that the ethanol has been added.
7. Wash Solution #2/3:  Add four volumes (201.6 ml) of ACS Grade 100% (200 proof) ethanol to the 50.4 ml of Wash Solution #2/3 Concentrate. Mix well. Place a check mark in the empty box on the label to indicate that the ethanol has been added. A precipitate will usually form in the bottle upon storage; this is excess EDTA falling out of solution. Leave these crystals in the bottle when removing wash solution for use.
8. Thoroughly clean the BSL-2 hood and place down fresh absorbent pads in the workspace where the extraction will take place.

Cell Lysis and RNA Extraction
1. Homogenize and lyse the cells using the Polytron Homogenizer.
2. Upon the completion homogenize and suspend in 5 ml of TRIzol Solution in a 30ml, round-bottom polypropylene tube. Shake all tubes thoroughly to ensure the homogenization of cell lysis and TRIzol solution.
3. Place the 30 ml tubes in a rack in the hood and allow them to incubate at room temp. for 10 minutes.
4. While the samples are incubating, set up the Promega Vac-Man vacuum manifold. Make sure that all ports have a new, clean stop-cock. Obtain a clean syringe filter (included with the kit) for each RNA sample and label one for each sample extraction number. Fasten each syringe filter to a stop-cock. Now obtain a clean, sterile 10ml syringe for each RNA sample. Remove each syringe from its individual packaging and attach them to the syringe filters on the manifold, leave the plungers in the syringes for the time being. Place a rubber stopper in the drainage port of the manifold. Put all of the stop-cocks that have a filter and syringe in the open position. Those with no filter and syringe need to remain shut throughout the procedure.
5. At the end of the 10 minute incubation period, add 1000ul of molecular grade Chloroform to each tube containing the cell lysis homogenate. Shake each tube vigorously for 15-20 seconds to ensure complete mixing of the chloroform and the homogenate. Caution: Chloroform is a known carcinogen. Always handle it in a hood with proper ventilation and dispose of it by the proper laboratory means.
6. Transfer the chloroform/homogenate from the 30 ml tubes to the clean pre-labeled 15ml conical tubes.
7. Spin the tubes at 12,000 x g (or 10,000 rpm) for 30 minutes at 4 degrees Celsius.
8. While the samples are spinning, set-up the custom-made sample elution vessel. Label a clean, sterile 15ml round-bottom tube for each RNA sample being extracted. Place the tubes in consecutive order in the interior rack of the elution vessel. Place the top on the vessel. The top of the vessel contains ports that the syringe filters will fit into. Any port that has no collection tube directly below it must be sealed with tape so that it is air tight.
9. Fill a 250ml glass beaker halfway with deionized water. Place the beaker on a hot plate. Obtain the Elution Solution from the RNAqueous kit and fill a 50ml conical with enough solution so that each sample extraction can receive 3ml of the Elution Solution (ex: if there are 10 samples, fill the tube with at least 30 ml of solution). Cap the tube and place it in the glass beaker. Turn the hot plate on high heat and bring the water in the beaker to a boil.
10. When the samples have finished spinning, remove the tubes from the centrifuge. Each tube should now contain 3 layers, an upper clear layer, a middle solid layer, and a lower pink layer. Bring the tubes back to the hood.

11. Remove the upper clear aqueous layer from each tube and pipette it into a clean, sterile, pre-labeled 15 ml conical tube. Be very careful to only pipette up the clear layer and none of the two lower layers. Place each tube with the aqueous layer in wet ice until this step is completed for all samples.

12. Add 1 volume of chilled 64% ethanol to the aqueous layer in the new tubes. Shake each tube vigorously for 15-20 seconds to ensure complete homogenization of the aqueous layer and the ethanol.

13. Remove the sample tubes to the bench top and turn on the vacuum supply. Remove the plungers from the syringes on the manifold and pour each sample into its corresponding syringe. Gently tough the vacuum hose to the vacuum port of the manifold and allow the sample solutions to be pulled slowly through the syringe filters. Be careful not to apply too much vacuum pressure to the manifold or the filters may rip, causing the RNA to be lost. When a sample has been pulled completely through the filter, close off its stop-cock. Continue pulling the samples through until all stop-cocks are closed.
14. Once all the samples have been pulled through their filters, open up all the stopcocks and apply additional vacuum pressure to the manifold. Watch the neck of the stop-cocks and you will see a foamy substance being pulled down. Continue applying gentle pressure until all of this foamy substance is pulled out.
15. Remove a 10ml serological pipette from its wrapper and add 6ml of Wash Solution # 1 to each syringe/filter set-up. Gently pull the wash solution through using the vacuum pressure and closing off the stop-cocks as you go. When all of the Wash Solution #1 has been pulled through each filter, open the stop-cocks up and apply gentle pressure to remove the foam.

16. Remove a 5ml serological pipette from its wrapper and add 4.20ml of Wash Solution #2/3 to each syringe/filter set-up. Gently pull the wash solution through using the vacuum pressure and closing off the stop-cocks as you go. When all of the Wash Solution #2/3 has been pulled through each filter, open the stop-cocks up and apply gentle pressure to remove the foam.

Now repeat step 16 a second time!

RNA Elution and Precipitation

1. Upon completing all of the wash steps, dispose of the syringes properly. Remove each syringe filter from the manifold and snap them firmly in place in the lid of the elution apparatus so that its corresponding collection tube will be directly below it when the lid is properly in place.

2. Drain the pull-through solution inside of the vacuum manifold into the flammable waste container in the chemical storage hood. Spray Envirocide inside of the manifold, fill it with water, and then leave it tipped up in the sink to drain. When the manifold is dry put it away.

3. Remove a clean, sterile 5ml syringe from its wrapper and attach one to each of the syringe filters now snapped into place on top of the sample elution apparatus. Remove the plungers from each syringe.

4. Make sure that the gasket of the elution apparatus is in place and that the seal between the lid and the chamber is air tight.
5. By now, the water in the glass beaker should be boiling and the elution solution in the 50 ml tube in the water should be at the proper temperature. Remove the tube from the water and add 1ml of Elution Solution to each of the 5ml syringes attached to the syringe filters.

6. Open the stop cock to the chamber and apply gentle vacuum pressure to the chamber so that the elution solution is pulled completely through each filter and into the corresponding collection tubes.

Now repeat step 6 2 more times so that each filter receives a total of three milliliters of Elution Solution.7. Remove the lid from the chamber and dispose of the syringe filters. At this point, the RNA should be in solution in the collection tubes.

8. Add 300ul of 3M Sodium Acetate, pH 5.2 to each collection tube.

9. Add 3ml of 100% Isopropanol to each collection tube.

10. Add 1ul of molecular grade Glycogen to each collection tube.

11. Cap each collection tube and invert them several times. As you invert the tubes, ripples should appear on the sides of the tubes. Continue inverting the tubes until these ripples have disappeared.
12. Place each collection tube in a rack and incubate the samples at -20 degrees Celsius for at least one hour. This incubation stage allows for the precipitation of the RNA.
13. After the hour incubation period, remove the samples from -20 and spin them at 12,000 x g (or 10,000 rpm) for 30 minutes at 4 degrees Celsius.

14. When the samples have finished spinning, a white pellet should be noticeable at the bottom of each tube.

15. Pour off the solution in each tube into a waste beaker being careful not to dislodge and lose the pellet. Dab the opening of each tube on a stack of Kim wipes or paper towels to remove any residual solution.

16. Allow the tubes to air dry for a minute and while this is happening, label a 1.5 ml Eppendorf tube for each sample.

17. Wash the samples by resuspending the pellets in the 15ml tubes with chilled 70% Ethanol. Pipette the mixture up and down several times to ensure that the pellet becomes fully resuspended.

18. Fill the tube holes in a heat block with RNase-free water. Set the heat block to 65 degrees Celsius and allow it to heat up.

19. Spin the samples in the Eppendorf tubes at 12,000 x g or (10,000 rpm) for 5 minutes at 4 degrees Celsius.

20. When the spin is complete, pour off the ethanol making sure not to dislodge and lose the pellet. Dab the mouths of the Eppendorf tubes on a stack of Kim wipes to remove any excess ethanol.

21. Use a clean sterile cotton-tipped applicator to remove any residual ethanol inside each tube. Make sure to use a new applicator for each sample and be careful not to make contact with the RNA pellet.

22. Discretion must now be taken when resuspending the pellets in RNase-free water. If the pellet is small (approximately the size of a pin-head) add 100ul of RNase-DNase-Free water. If the pellets are larger, anywhere from 110 – 200ul of water may be used.

23. Once you have added the water to each sample place the tubes with the caps open in the heat block. Leave the tubes on the heat block for about 1 minute as this will aid in removing any left over ethanol.24. After a minute, resuspend the pellets in the water by gently pipetting up and down several times.
25. Keep all samples on wet ice until needed for Quantitation and QC.