The often exacting process of isolating intact total RNA from tissue becomes even more difficult when processing certain problematic tissues. Fibrous tissues and tissues rich in protein, DNA and nucleases present distinct challenges for total RNA isolation. Some of the demanding tissues requiring more manipulation and fine-tuning during the RNA isolation procedure are heart, brain, thymus and spleen. Here we address problems we have encountered, and offer troubleshooting techniques to help overcome problems associated with isolating total RNA from difficult tissues. The tips provided are based on RNA isolation by a guanidinium thiocyanate/acid phenol:chloroform extraction method (e.g. Invitrogen Ambion's ToTALLY RNA Kit). Note that many of these techniques can be used with other RNA isolation protocols as well.
Heart and Skeletal Muscle: Fibrous Tissues
For fibrous tissues such as rat and mouse heart and skeletal muscle, the most difficult step in the isolation process can be complete disruption of all the cells when preparing tissue homogenates. Due to low cell density and the polynucleate nature of muscle tissue, the yield of total RNA is typically low; therefore, making the most of the tissue on hand is critical. Preparation for homogenization should be carried out on dry ice, under liquid nitrogen. Pulverizing the tissue into a powder while keeping the tissue completely frozen is key to isolating intact total RNA. Large chunks of fibrous tissue are difficult to homogenize completely and can result in degraded RNA and very low yield. Unfortunately, the signs of complication occur only at the end of the isolation process, during calculation of yield and visualization of the total RNA on a gel. Therefore, care should be taken during the initial preparation of the homogenate to ensure intact total RNA.
Making Difficult Isolations Easy
|Tissue characteristics||Symptoms||Representative tissues||Procedure modifications|
|Fibrous tissues||Difficult to homogenize||Thorough disruption: Freeze tissue and grind|
|Nucliec add-rich||Viscous lyate, incomplete phase seperation, precipitation on addition of isopropanol||Dilute lysate|
Do additional extraction
|Protein and lipid-rich||White flocculent material in aqueous phase of extraction||Dilute lysate|
Modify extraction (more CHCI3)
|Nuclease-rich||Degraded RNA||Efficient disruption|
Freeze tissues and grind
Brain and Plant Tissues: Protein and Lipid-rich Tissues
Isolating total RNA from rat or mouse brain can be very rewarding because of the large yields recovered once troubleshooting techniques have been implemented. Brain and plant tissues are rich in lipids, which can complicate the RNA extraction process, making it difficult to get a clean separation of RNA. An obvious sign of trouble occurs once the brain or plant homogenate has been extracted with phenol:chloroform:IAA. White flocculent material will make up most of the volume of the aqueous phase after centrifugation. This white material likely contains lipids and does not form a tight interface. To remedy the situation, add one-tenth volume chloroform:IAA (i.e, 0.1 x the sum of aqueous and organic volumes), mix well, and recentrifuge. To increase yield, back-extract the organic phase and re-extract the aqueous phase with phenol:chloroform. Alternatively, remix the aqueous and organic phases, add more lysis solution, effectively diluting the protein and lipids, and re-extract with phenol:chloroform:IAA. It has also been suggested that extracting a plant tissue lysate with chloroform first, before proceeding with RNA isolation, prevents the white, flocculent material from forming.
An option for plant tissue involves the use of polyvinylpyrrolidone (PVP) in the lysis step prior to the organic extractions. PVP complexes with polysaccharide and polyphenol compounds commonly found in plants. The complexed material is centrifuged out of the lysate, and the lysate is then processed according to protocol. This step prevents carryover of contaminants that may inhibit downstream applications. Ambion's Plant RNA Isolation Aid is a solution of PVP that can be added to plant tissue lysates for removal of such contaminants.
Rat Spleen and Thymus: Nucleic Acid and Nuclease Rich Tissues
Rat spleen and thymus are high in nucleases and nucleic acids. Efficient homogenization is critical for reducing the effects of nucleases found in the tissues. Pulverization of the spleen and thymus into small pieces on dry ice, under liquid nitrogen, allows for quick homogenization in lysis solution, which inactivates nucleases. The phenol extraction step of the tissue lysates can also present problems. The high DNA and RNA content of these tissues causes the homogenates to be unusually viscous. Extraction of such viscous homogenates sometimes results in incomplete phase separation. Adding more lysis solution and/or re-extracting with phenol:chloroform:IAA will help alleviate this problem. Also, multiple phenol:chloroform:IAA extractions can be performed to ensure the partitioning of DNA into the organic phase during the acid phenol extractions of the RNA isolation procedure. If a white precipitate forms immediately upon the addition of isopropanol, post-acid phenol extraction, this is a sign that DNA contamination is still present. Centrifuge the precipitate, resuspend the pellet in nuclease-free water, and extract with phenol:chloroform:IAA until this quick-forming white precipitate no longer occurs. High yields of intact RNA can be achieved once abundant DNA and nucleases are brought under control.
RNAlater: Making RNA Isolation From Difficult Tissues Less Difficult
With any of the tissues listed above, one of the challenges is to halt RNase activity after the tissue is harvested. This is often achieved by rapidly cutting the tissue into manageable pieces, and placing the pieces into liquid nitrogen. "Snap-freezing" halts all RNase activity in the tissue. While frozen solid, the tissue pieces are ground into a powder with a mortar and pestle. The powder is then processed according to the RNA isolation protocol. However, RNA can still be degraded if the dissection takes too long or the frozen tissue thaws.
Invitrogen RNAlater, an aqueous, non-toxic tissue and cell storage reagent, stabilizes and protects cellular RNA in intact, unfrozen tissue and cell samples. The tissue is simply dropped into 5 volumes of RNAlater. The reagent permeates the tissue immediately and inactivates RNases. RNAlater, therefore, obviates the need to use liquid nitrogen, which is frequently inconvenient and, in certain settings (e.g. fieldwork), not a realistic option for rapid preservation of tissues. Tissue samples treated with RNAlater can be stored for a day at 37°C, a week at 25°C, a month at 4°C, and indefinitely at -20°C. RNA can be isolated with standard one-step phenol extraction methods or glass-binding methods, as well as with methods that use oligo d(T) selection of mRNA. Tissue is simply removed from RNAlater and processed like fresh tissue in the RNA isolation lysis solution. There is no need to freeze and grind the tissue using a mortar and pestle, though freezing and grinding can be done if desired. (Note: RNAlater may not be suitable for some plant and bacterial samples.)