TRIzol reagent protocol (Life Technologies, Inc., Gaithersburg, MD)

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In summary, the individual used the TRIzol reagent protocol for RNA isolation from a human tumor and is now looking for a kit to extract mRNA from the total RNA suspension. They have found that Quiagen kits are only suitable for animal tissues and not human suspension. They were given a link to a SuperArray kit that may work for their purposes. The individual also asked about the step of incubating the RNA sample at 55 to 60°C for 10 minutes, which is just to get it into solution faster, and was advised to be careful with air-drying the isolated RNA to avoid contamination.
  • #1
sobored
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I used TRIzol reagent protocol (Life Technologies, Inc., Gaithersburg, MD) for isolation of my human tumour total RNA and i wonder if there are any kits i can use to isolate mRNA from this suspension? I have looked in Qiagen and they only have kits for mRNA isolation from animal tissues and not human suspension. :frown:



THanks for any help.
 
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  • #2
:rolleyes: Humans are animals.

I'm not entirely sure what step you're stuck at. The protocol that comes with Trizol is pretty good and straightforward.
 
  • #3
I am looking for a kit to extract mRNA from this total RNA suspension. The Quiagen kits are only for extracting mRNA directly from the tissues and not from total RNA suspension. :frown:
 
  • #4
Do you mean something like this?

http://www.superarray.com/newsletter/RNA.html
Enriched mRNA samples are also suitable for microarray gene expression profiling experiments. SuperArray recently developed the ArrayGrade mRNA Purification Kit that combines the oligo-dT affinity purification technique with a magnetic microparticle technology. The Oligo-dT14 is immobilized on 1 micro, super-paramagentic microparticles. The oligo-dT microparticles are uniform, colloidally stable and non-porous spheres that offer high surface area and can remain in suspension to facilitate the interaction between the poly (A) tail and oligo-dT.
 
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  • #5
Thanks for that link.

Since you have read the protocol of Trizol. Do you know why i should incubate my RNA sample for 10 minutes at 55 to 60°C? This would degrade my RNA and how come i use it for further working?

The protocol says; "5. REDISSOLVING THE RNA
At the end of the procedure, briefly dry the RNA pellet (air-dry or vacuum-dry for 5-10 minutes). Do not dry the RNA by centrifugation under vacuum. It is important not to let the RNA pellet dry completely as this will greatly decrease its solubility. Partially dissolved RNA samples have an A260/280 ratio < 1.6. Dissolve RNA in RNase-free water or 0.5% SDS solution by passing the solution a few times through a pipette tip, and incubating for 10 minutes at 55 to 60°C. (Avoid SDS when RNA will be used in subsequent enzymatic reactions.) RNA can also be redissolved in 100% formamide (deionized) and stored at -70°C."

http://www.invitrogen.com/content/sfs/manuals/15596026.pdf


Thanks.
 
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  • #6
As far as I know, that's just to get it into solution faster.
 
  • #7
The step at 55C is to get the RNA into solution faster. It is the same idea has with DNA. You will not see a significant degradation of RNA if you RNA was carefully resuspended with any material that is RNase free.

You may skip this step if you see that your RNA went into solution quickly after you added your dilutent.
 
  • #8
The protocol says that i have to air-dry the isolated RNA. Would not this contaminate the sample with RNase and damage my RNA sample?


Thanks.
 
  • #9
Don't air dry the tube right side up. Tube upside down or on its side will reduce the odds of contamination.
 

Related to TRIzol reagent protocol (Life Technologies, Inc., Gaithersburg, MD)

1. What is the purpose of using TRIzol reagent?

TRIzol reagent is commonly used in molecular biology to isolate and purify RNA, DNA, and protein from a variety of biological samples. It is designed to efficiently disrupt cells and inactivate nucleases, allowing for the isolation of high-quality nucleic acids for downstream applications.

2. How does TRIzol reagent work?

TRIzol reagent is a monophasic solution of phenol and guanidine isothiocyanate, which efficiently lyses cells and denatures proteins. The guanidine isothiocyanate denatures proteins and inactivates nucleases, while the phenol disrupts the cell membrane and denatures remaining proteins. This results in the separation of RNA, DNA, and protein into distinct phases, allowing for their isolation.

3. What is the recommended protocol for using TRIzol reagent?

The recommended protocol for using TRIzol reagent involves three main steps: sample lysis, phase separation, and precipitation. First, the sample is lysed and homogenized in TRIzol reagent. Next, chloroform is added to the lysate and the mixture is centrifuged, resulting in the separation of the sample into an aqueous phase (containing RNA), an organic phase (containing DNA), and an interphase (containing proteins). Finally, the RNA or DNA can be precipitated from the aqueous phase using isopropanol, washed with ethanol, and resuspended in an appropriate buffer for downstream applications.

4. How do I store TRIzol reagent?

TRIzol reagent should be stored at room temperature and protected from light. It is important to avoid exposure to extreme temperatures, as this can affect the integrity of the reagent. Additionally, TRIzol reagent should not be frozen, as this can cause phase separation and result in loss of activity.

5. Can TRIzol reagent be used for all types of biological samples?

TRIzol reagent has been successfully used for a wide range of biological samples, including mammalian tissues, cells, bacteria, yeast, and plant tissues. However, some samples may require additional steps or modifications to the protocol to optimize RNA or DNA isolation. It is always recommended to perform a pilot experiment to determine the most suitable protocol for a specific sample type.

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