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Having difficulties with your experiment?
We are dedicated to your success. Get back on track. View our expert recommendations for commonly encountered problem scenarios.
View the relevant questions below:
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Heat the solution to 37°C for 15 minutes and agitate to redissolve it.
These are the top 10 ways to improve your RNA isolation results:
1) Immediately inactivate endogenous, intracellular RNases.
2) Use proper cell or tissue storage conditions.
3) Thoroughly homogenize samples.
4) Pretreat homogenate before RNA isolation to remove interfering compounds.
5) Choose the best RNA isolation method for your sample.
6) Include a DNase treatment.
7) Reduce exposure to environmental RNases.
8) Precipitate appropriately for the downstream application.
9) Resupend the RNA properly.
10) Store the RNA properly after isolation.
Read more about each suggestion here.
There are two methods to remove insoluble material:
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:
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:
A portion of the interphase may have been removed with the aqueous phase after the initial separation. This can occur for several reasons.
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:
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:
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:
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.
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:
Perform a DNase I digestion with the total RNA sample to remove any genomic DNA contamination before isolation of RNA using the RiboMinus™ kit.
Yes, it is available in 1 mL aliquots (Cat. No. 4402960).
Please review the following possibilities and suggestions:
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:
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.
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