Yes, as long as the antibody used binds to the target protein in its denatured form.

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We recommend using our Purelink RNA Mini Kit (Cat. No. 12183025) or TRIzol Reagent (Cat. No. 15596026) to isolate your RNA. Oligo(dT) selection for mRNA is typically not necessary, although it may improve the yield of specific cDNAs.

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Most likely the sample was not immediately processed or frozen immediately. You should process samples immediately or freeze the samples. Store RNA samples at -60 degrees C to -70 degrees C. Store DNA and protein samples at -20 degrees C.

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- 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.

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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.

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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.

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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.

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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 degrees 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 (https://www.thermofisher.com/us/en/home/life-science/dna-rna-purification-analysis/rna-extraction/rna-sample-extraction/working-with-ffpe-samples.html).

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A lower yield can indicate that the samples may have been incompletely homogenized or lysed. You could decrease the amount of starting material or you could cut the tissue samples into smaller pieces and ensure that the tissue is completely immersed in TRIzol Reagent. Alternatively, the pellet may have been incompletely solubilized. If this is suspected, heat the sample to 50 degrees C to 60 degrees C and pipette the sample repeatedly.

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- 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 degrees 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 degrees 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 degrees 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 approximately 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.

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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 degrees 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.

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Most likely the interphase/organic phase is pipetted up with the aqueous phase. Do not attempt to draw off the entire aqueous layer after phase separation.

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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 degrees 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 degrees C instead of -70 degrees 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.

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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.

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Degraded RNA can cause an increased absorbance at 260 nm.

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