RNA Sample Collection, Protection, and Isolation Support—Troubleshooting

Heat the solution to 37°C for 15 minutes and agitate to redissolve it.

There are two methods to remove insoluble material:

1. For RNA isolation only: If a lot of insoluble material exists after homogenization and a 5 minute room temperature incubation, remove it by centrifugation at 12,000 x g for 10 minutes at 4°C before adding chloroform (a clear supernatant and jelly-like pellet should be seen). Remove the supernatant and proceed to the next step. Note: This should not be done if subsequent DNA isolation is planned.
2. RNA and DNA isolation: If a lot of insoluble material exists after homogenization and a 5 minute room temperature incubation, the homogenate can be passed through a polypropylene mesh to remove insoluble material that may interfere with precipitation of DNA.

If isopropanol is inadvertently added at this step instead of chloroform, add more isopropanol to precipitate everything, then resuspend the pellet in TRIzol® Reagent and use the protocol as specified. RNA yields will be compromised, but it may be possible to obtain a product in downstream RT-PCR. Here is the detailed protocol:

1. Add more isopropanol so that the total volume of isopropanol equals the volume of TRIzol® Reagent used. Centrifuge at 7,500 x g for 10 minutes at 4°C.
2. Pour off supernatant; allow relatively compacted pellet to air-dry (doesn't have to be completely dry, just reduce the volume of ispropanol).
3. Estimate the size of the pellet in microliters; add at least 15–20 volumes of TRIzol® Reagent (e.g., for a 100 μl pellet, add at least 1.5 mL TRIzol® Reagent).
4. Break the pellet up well (you may have to use a hand-held homogenizer). Store the solution for 10–15 minutes at room temperature; every 5 minutes or so, shake it by hand to make certain it is well dispersed.
5. Proceed with the TRIzol® protocol as written (i.e., add chloroform). Results will not be optimal, but it may be possible to obtain a product in RT-PCR.
   Alternatively, add 2 additional volumes of TRIzol® Reagent to make up for the added volume, and then add more chloroform based on the total volume of TRIzol® Reagent. This method is quicker, but will yield more DNA contamination of the RNA.

If a larger amount of chloroform than needed was inadvertently added, you should add more TRIzol® Reagent so that a ratio of 0.2 mL chloroform: 1 mL TRIzol® Reagent is maintained. If too much chloroform is added, this will drive the DNA, and eventually the protein, into the aqueous phase.

Please review the following causes for low yield of RNA/degraded RNA:

• The RNA may have been concentrated with a SpeedVac™ system or lyophilized after the last ethanol precipitation. RNA that has been dried completely has decreased solubility. Additionally, if excess centrifugation speeds (higher than 12,000 x g) were used, it is harder to solubilize RNA/DNA.
• The RNA pellet may not be completely solubilized. To increase the rate of solubilization, pipette repeatedly in SDS solution or DEPC-treated water, then heat to 50–60°C. The sample may also have been rich in polysaccharides or proteoglycans. If so, the isopropanol precipitation step should be done with 0.25 volumes of isopropanol and 0.25 volumes of a high salt solution.
• Cells were washed prior to the addition of TRIzol® Reagent. Washing cells before the addition of TRIzol® Reagent increases the possibility of mRNA degradation.
• The sample was not fully homogenized.
• The tissue was not IMMEDIATELY processed or frozen after removal from the animal or other source.
• The tissue was not completely disrupted; if a centrifugation is done prior to adding chloroform, there should be a white mucus-like pellet. If there is a tan-colored precipitate, this is indicative that not all of the cells have been lysed.
• If a mortar and pestle was used to powder the tissue, RNA and DNA may have stuck nonspecifically to the mortar and pestle. It may be better to use a glass homogenizer and Teflon® pestle; add TRIzol® Reagent to the homogenizer, then add frozen tissue and homogenize.
• RNA may have been stored after isolation at –20°C instead of –70°C.
• Tissue culture cells were disrupted by trypsin.
• Homogenizing for too long and too continuously in a small volume (e.g., 1 mL) may cause heating of the sample; this may result in degradation of the RNA in the tissue. Samples should be cooled during homogenization, and homogenization should be done in on-off cycles (as opposed to continuously).
• The OD reading may vary due to the solution the sample is stored in AND what it was diluted in prior to quantitation. This can lead to apparently low yields.
• Excess RNAlater® Stabilization Solution (>0.05 mL) will reduce RNA recovery and cause problems with phase separation.

A portion of the interphase may have been removed with the aqueous phase after the initial separation. This can occur for several reasons.

• An insufficient amount of TRIzol® Reagent was added to the sample. In general, 1 mL of TRIzol® Reagent should be used for every 0.05 g of tissue or every 10 cm2 dish.
• The original sample may have had traces of other organic material to begin with (ethanol, DMSO, etc.).
• RNA should be treated with amplification grade DNase I prior to RT-PCR.
• There may have been insolubles after the first homogenization that were not removed by centrifugation before chloroform extraction.
• If the DNA contamination was detected in RNA isolated from cells after transfection with a plasmid, not all of the plasmid DNA may have partitioned into the interphase/organic phase once the chloroform was added to the TRIzol® Reagent. If RT-PCR is being used to assay gene expression from the transfected plasmid, a DNase I treatment will be needed.

• The sample was homogenized in too small a volume of TRIzol® Reagent.
• The sample was not stored at room temperature for 5 minutes after homogenization. (This may result in nuclear proteins not being dissociated).
• The final RNA pellet was not fully dissolved. This may be the case if the RNA pellet was overdried (if the pellet is clear and not white, this indicates overdrying). To get the pellet to dissolve completely, heat to 55–60°C for 10–15 minutes and repeatedly pipette.
• There may be phenol contamination. This may occur if samples were centrifuged at room temperature instead of 4°C; phenol is more soluble in the aqueous phase at room temperature. If absorbance is seen at 270 nm (due to phenol), the sample can be ethanol precipitated to remove the residual phenol.
• Guanidine absorbs around 240 nm. Phenol has two peaks: one around 275 nm, the other a broad peak ranging from below 220 to around 240 nm. If a very large peak is observed in that range, we recommend that you precipitate and wash again. To prevent this, we also recommend doing the phase separation after addition of chloroform at 4°C.
• Residual chloroform may be present; reprecipitate.
• In some samples dissolved in water, the ratio may be low due to the acidity of the water or the low ion content in the water. The ratios may go up if the sample is dissolved in TE and the spectrophotometer is zeroed with TE buffer (or 1–3 mM Na₂HPO₄, pH ~8.0). The molar extinction coefficient of the nucleotides is given at neutral pH, suggesting that the absorbance at 260 nm would be highest at neutral pH.
• A variation in A260/A280 ratios with different spectrophotometers (it seems that the A280 value varies depending on the way the unit was "blanked" at 280 nm after being "blanked" at 260 nm). In this case, we suggest using a different spectrophotometer.

You can add glycogen to your sample, which can help improve yield and remains with the RNA (glycogen is water soluble). Polyacrylamide can also be used as a carrier to precipitate small amounts of RNA. Alternatively, you can also use salmon sperm DNA. It should be added during the precipitation of the aqueous phase.

If a sample is known to have a high content of proteoglycans and/or polysaccharides (such as rat liver, rat aorta, plants), the following modification of the RNA precipitation step should remove these contaminating compounds from the isolated RNA:

Add 0.25 mL of isopropanol to the aqueous phase followed by 0.25 mL of a high-salt precipitation solution (0.8 M sodium citrate and 1.2 M NaCl; no pH adjustment necessary) per 1 mL of TRIzol® Reagent used for homogenization. Mix the resulting solution, centrifuge, and proceed with isolation as described in the protocol.

This modified precipitation effectively precipitates RNA and maintains proteoglycans and polysaccharides in a soluble form. To isolate pure RNA from plant material containing a very high level of polysaccharides, the modified precipitation should be combined with an additional centrifugation of the initial homogenate. In general, we do not recommend high-salt precipitation if polysaccharide or proteoglycan contamination is not a concern, since it is an extra step and there is otherwise no significant advantage to adding this step. When purifying an RNA sample where polysaccharide or proteoglycan contamination is not an issue, in general, the total RNA yield will be same with or without the high salt. There may be small changes in the RNA profile reflected by slightly decreased amounts of tRNA. The high-salt precipitation reduces tRNA in the sample.

These are our recommendations:

1. Upstream tissue procurement and tissue specimen preparation—if possible, fix tissues within one hour of surgical resection. Extensive degradation of RNA can occur before completion of the fixation process. The optimal fixation time is 12–24 hours, using neutral-buffered formalin or paraformaldehyde. Fixed tissues should be thoroughly dehydrated prior to the embedding process.
2. Block storage—storage of blocks without cut faces, when possible, prevents ongoing damage from exposure to atmospheric oxygen, water, and other environmental factors such as light and infestation (fungus, insects, etc.).
3. Choice of tissue type, size, and amount being used for RNA isolation—the recommended tissue thickness is 10–20 µm. The number of sections used is determined by the tissue type (which impacts cell density) and surface area (recommended size: 50–300 mm2). Excess starting material can cause filter clogging, resulting in poor yield.
4. Avoid using an excessive amount of paraffin for embedding tissues—when possible, excess paraffin should be trimmed away prior to starting the purification protocol. For xylene-based purification methods, two xylene treatments at room temperature should be sufficient for complete deparaffinization. If desired, you can perform a more rigorous 37–55°C treatment for up to 30 minutes. After the xylene deparaffinization, it is crucial that the 100% ethanol is completely removed and the pellets are dry after the two 100% ethanol washes. The magnetic bead method employs novel chemistries to deal with the paraffin that limits input to 20 µm sections.
   Read more about RNA isolation from FFPE tissues here.

• This is common with skin samples. It is assumed that there is fat in these samples, and the fat micelles float during the centrifugation. In skin samples, the micelles pick up melanin pigment and cause the aqueous phase to appear colored. Fat micelles may also pick up pigment from the TRIzol® Reagent itself and cause a pinkish color. If a sample is thought to contain fat, the sample homogenate in TRIzol® Reagent may be centrifuged prior to addition of chloroform. The fat will appear as a clear layer at the top of the supernatant; this should be pipetted off and discarded.
• If a sample contains a lot of blood, the aqueous phase may appear cloudy and/or yellowish (this may be due to iron in the hemoglobin). If the centrifuge used is not cold, the organic phase will be a deeper maroon color; some of this color may come into the aqueous phase and cause it to appear orange or yellow.
• A pinkish aqueous phase may also be caused by overdilution of the sample (i.e., a sample to TRIzol® Reagent ratio > 1:10), as well as too much salt or protein in the sample. This can cause premature phase separation, which can be remedied by adding a bit more TRIzol® Reagent to the sample. If the RNA is isolated from a pinkish aqueous phase, chances are that it will be contaminated with DNA.

This is most likely polysaccharides or cell membranes; DNA should be in the interphase. In samples containing blood (e.g., liver), a red viscous layer may be visible on top of the pellet. This is most likely due to blood products and should not be carried over with the supernatant.

Degraded RNA can cause an increased absorbance at 260 nm.

To prevent filter clogging with viscous samples or samples containing a large amount of tissue, perform a clarifying centrifugatoin at 10,000–15,000 x g and remove to a fresh tube prior to adding ethanol to the lysis/binding solution.

Poor quality/poor purity RNA is typically due to the following:

• Partially degraded RNA: Use fresh tissue or cells, or use RNAlater® Stabilization Solution if tissue cannot be frozen immediately.
• DNA contamination: If the sample contains organic solvents, strong buffers, or an alkaline solution, we recommend performing phase separation at 8°C.
• A low A260/A280 ratio (<1.6): This indicates that an insufficient amount of the TRI Reagent® was used. Incubation of the homogenate at room temperature for 5 minutes can help nucleoproteins dissociate from RNA. Phenol contamination could also cause a low absorbance ratio.

Incomplete homogenization or dispersal of precipitate after ethanol addition can lead to clogging of the RNA spin cartridge. Clear the homogenate and remove any particulate or viscous material by centrifugation. Completely disperse any precipitate that forms after adding ethanol to the homogenate. Load only the supernatant onto the RNA spin cartridge to avoid clogging.

Low RNA yield can occur due to the following:

• Incomplete lysis and homogenization: ensure that 10 µL of 2-mercaptoethanol was added per milliliter of lysis buffer, perform all steps at room temperature, decrease the amount of starting material used, use proper homogenization methods, and/or cut tissue samples into smaller pieces to ensure complete tissue immersion in the lysis buffer.
• Poor quality starting material: use fresh samples and process immediately after collection.
• Ethanol may not have been added to Wash Buffer II.
• Incorrect elution conditions may have been used: Add RNase-free water and incubate for 1 minute before centrifugation, following the recommendations for elution in the manual. You can also perform a second elution step to recover more RNA.

The RNA could have been contaminated with RNase. Ensure that you are using RNase-free equipment and change gloves frequently. Improper handling can also result in RNA degradation. Ensure samples are processed immediately, and that the lysis is performed quickly after adding the lysis buffer. Lastly, tissues rich in RNase (such as rat pancreas) may require the addition of RNase inhibitors or inactivators to protect the RNA from degradation, or a larger volume of lysis buffer.

The presence of ethanol or salt in the purified RNA can inhibit downstream enzymatic reactions. Ensure that you are using the correct order of wash buffers in the kit for washing, and that Wash Buffer II is discarded in the flow-through. Place the spin cartridge into the wash tube and centrifuge the spin cartridge at maximum speed for 2–3 minutes to completely dry the cartridge.

• Precipitation of miRNA: Follow the instructions for the mirVANA™ miRNA Isolation Kit or the mirVANA™ PARIS™ RNA and Native Protein Purification Kit.
• Double phenol/guanidium (chaotropic reagent) extraction: Disrupt tissue with phenol/guanidine, then extract with phenol, centrifuge, remove the supernatant, and precipitate the RNA.
• Glass-fiber filter: Disrupt the tissue in a chaotropic agent, bind the RNA to a glass-fiber filter, then wash off unbound DNA/protein/salt/nucleotides and elute the RNA.

You would have to separate the virus from the cells prior to isolation to avoid contamination of the sample with cellular nucleic acids.

Low RNA content or a loss of the pellet during ethanol precipitation could lead to a low RNA yield. Additionally, various tissues have different RNA content, and the yield is dependent on the sample.

There are several possibilities:

• Too much total RNA may have been used: Please check the amount of total RNA you are using (we recommend between 2 and 10 µg total RNA).
• A low amount of magnetic beads or probe was used: Use the recommended amount of probe or beads.
• Improper handling or drying of the beads may have occurred: Follow the recommended guidelines for washing/mixing beads; do not allow the beads to dry.

Perform a DNase I digestion with the total RNA sample to remove any genomic DNA contamination before isolation of RNA using the RiboMinus™ kit.

1. Ensure that all medium is removed from the wells.
2. Wash with an equal volume of room temperature 1X PBS after the medium is removed.
3. Ensure that the reaction happens at room temperature (the lysis reaction may not reach room temperature if the plate is on ice, if the plate was quickly moved to the bench, or if a cold lysis solution was added).
4. Warm lysis solution to room temperature before adding to cells.
5. Allow the lysis reaction to proceed for 8 minutes at 25°C.

Yes, it is available in 1 mL aliquots (Cat. No. 4402960).

Please review the following possibilities and suggestions:

• A problem with adding or mixing the Stop Solution: ensure that the Stop Solution was added directly to the lysate, as components of the Lysis Solution may inhibit RT-PCR if not fully inactivated.
• The RNA was degraded: keep cells in PBS on ice before starting the cell lysis procedure.
• RNase in the sample was not completely inactivated: Too many cells could have been used or too much PBS left on the cells, diluting the lysis solution.
• The lysates sat too long before going to room temperature: Do not allow lysates to sit longer than 20 minutes at room temperature once the Stop Solution has been added.
• The sample does not contain the target RNA: Verify that the procedure is working by using the XenoRNA™ Control in the sample. Also check that your PCR primers can amplify your target under the PCR conditions you are using.

PCR products in the no-template PCR control indicate that the sample is contaminated with DNA. More stringent steps need to be taken to control contamination.

If PCR products are seen in the minus-RT control reaction, but not in the no-template control, it indicates that genomic DNA remains in the sample and that genomic DNA was amplified in real-time PCR. Please follow the suggestions below:

• Ensure the DNase I is mixed thoroughly into the Lysis Solution.
• Use fewer cells per lysis reaction.
• Lyse cells using Lysis Solution that is at room temperature, and make sure that the lysis reaction occurs at room temperature.

You can also try increasing the incubation time of the lysis reaction to 8 minutes and/or using Lysis Solution that has been warmed up to 25°C for cell lysis.