RNA Isolation from Mouse Pancreas: A Ribonuclease-rich Tissue

The overall goal of this procedure is to isolate RNA with high integrity from the Ribonuclease ridge mouse pancreas. This is accomplished by quickly removing the pancreas from the carcass of a euthanized mouse. Next, the pancreas has submerged in lysis reagent, and the tissue is homogenized.

Following centrifugation, the RNA is isolated from the supernatant using standard protocols and RNAs inhibitor solution is added to the final RNA solution. The final step is to carefully clean the homogenizer blades by removing any chunks of tissue caught in the blades, and then performing a series of ethanol and water washes. Ultimately, this procedure allows for the isolation of RNA for mouse pancreas with an RNA integrity number greater than seven to be used for routine gene expression analysis.

My name's Tom Schmid and I've been working with RNA for over 20 years, and in all this time, I haven't experienced anything quite as difficult as isolating RNA with high integrity for mouse pancreas. The technique we're gonna show you is has been developed for mouse pancreas, but really there's no reason it couldn't be used for a variety of other tissues, including tissues from human cells, cell lines, or other tissues from mouse, including rib nuclease rich tissues such as the spleen. Using the suggestions in this protocol will allow you to isolate RNA with high integrity from mouse pancreas.

That could be used for many downstream applications such as QPCR, gene expression arrays, or RNA-Seq. Ola Elgamal. A senior grad student in my lab, will be demonstrating this technique After euthanizing the mouse as described in the text protocol.

Secure the carcass by piercing each of the animals four paws into a flat surface using 25 gauge hypodermic needles using sterile surgical instruments. Cut the midsection of the mouse open and quickly remove the pancreas from the mouse. Use the forceps to hold the pancreas and cut it away from the spleen and small intestine using spring scissors.

Be careful not to stretch the pancreas during the removal from the mouse. Disruption of the tissue could release ribonuclease. Place the entire pancreas into a 50 cubic centimeter tube holding eight milliliters of ice cold lysis reagent.

Cap the tube and briefly shake to immerse the tissue in the reagent and immediately proceed to the next step without delay. Next, homogenize the pancreas For five seconds. It is important to press the homogenizer blades to the bottom of the centrifuge tube to allow the tissue to be pulled into the blades for efficient homogenization.

To remove undigested tissue transfer two milliliters of lysis reagent containing the lysed pancreas to a two milliliter micro centrifuge tube. Collect two micro centrifuge tubes of homogenate per isolation centrifuge at four degrees Celsius, 12, 000 times G for five minutes to pellet the undigested tissue. This is a critical step as carryover of undigested tissue into subsequent solutions of RNA will likely contaminate the sample with ribonuclease.

Clean the homogenizer blades by placing them in 10 milliliters of 70%ethanol. Activate the homogenizer for about 20 seconds to remove any chunks of tissue that may be caught in the blades. Then use a 200 microliter pipette tip to remove any pieces that remain in the homogenizer blades.

Clean the homogenizer blades again in water general RNA syn activator and 70%ethanol. Finally, dry the homogenizer shaft with a clean Kim wipe. Transfer one milliliter of the SNA into a two milliliter micro centrifuge tube.

Place all tubes containing homogenate on wet ice while proceeding to the next step. Proceed with the RNA isolation immediately and store the replicate tube at minus 80 degrees Celsius for future use. Dispose of the remaining lysis reagent into an appropriate chemical waste receptacle.

The following section uses an RNA purification kit. The advantage of the purification kit is that it will isolate total RNA, including small RNAs. Begin isolation by adding 200 microliters of chloroform to the homogenate solution.

Cap the tube and shake vigorously by hand for 15 seconds. Then let the tube stand at room temperature for two to three minutes following centrifugation for 15 minutes at 12, 000 times G and four degrees Celsius. Transfer the upper aqueous layer into a micro centrifuge tube.

Add 1.5 volumes of isopropanol and mix thoroughly by pipetting up and down several times. Transfer up to 700 microliters of the sample into a spin column that is placed in a two milliliter collection tube. Close the lid and centrifuge at 8, 000 times G for 15 seconds at room temperature and then discard the flow through.

Next, add 700 microliters of buffer RWT to the spin column and centrifuge. As before, discard the flow through pipette 500 microliters of buffer RPE into the spin column and centrifuge again to wash the column. After discarding the flow through, add another 500 microliters of buffer RPE to the spin column centrifuge for two minutes at 8, 000 times G.To dry the column, transfer the spin column to a 1.5 milliliter micro centrifuge tube pipette 100 microliters of rib nucleus free water directly onto the spin column membrane centrifuge for one minute at 8, 000 times G to elute, the RNA.

Then add one microliter of RNAs inhibitor to the RNA solution and proceed to determine the RNA integrity using capillary electrophoresis analysis as described in the text protocol comparison of the RNA integrity number or RIN of RNA isolated from mouse pancreas reveals a direct correlation between the volume of lysis reagent used in the homogenization and RNA integrity. The results indicate that eight milliliters of lysis reagent should be used In this protocol, ribonuclease inhibitor was added to the aqueous solution of RNA and RNA integrity was compared upon freeze thaw. The RIN for the solution of RNA with inhibitor was 7.3 plus or minus 0.15 compared to 2.9 plus or minus 0.65 for the solution that did not contain inhibitor to examine the possibility that repeated freeze thawing will denature the ribonuclease inhibitor rendering it ineffective.

A solution of RNA containing ribonuclease inhibitor was tested. The RNA solutions that were frozen and thawed up to five times still produced a RIN of seven or higher. While those solutions that were frozen and thawed six times or higher produced a RIN below seven random prime, CDNA was synthesized from the RNA and the expression of three housekeeping genes was measured by QPCR.

Using standard techniques, the data validates successful use of mouse pancreatic RNA in a standard molecular biology technique. Once properly trained grad students and postdocs should be able to complete this technique in about 30 minutes. And I just wanna tell you a little bit about how we're using it.

We're studying transgenic mouse models of pancreatic cancer and we're using this technique to isolate RNA. And then when we get the RNA, we're going to study the gene expression of micro RNA in these animals.