What is the best method for RNA isolation from tissue samples?

I think the first thing to check is that you are running the gel properly. Are you using fresh buffer and only running for 10-15 minutes? If you have access to a Bioanalyzer then try running some of your samples through that to double check if your RNA integrity is actually at fault.

The tips I have for RNA extraction is to work quickly, put samples as soon as possible into solutions which kill/inhibit RNases (normally the solutions in which you mince/homogonize, eg Trizol), and keep things cold.

Gaurav Chhetri

it seems your RNA has degraded you can try these precaution,

First clean with detergent and wipe with alcohol then bake all the equipment you use for RNA isolation i.e. mortar pestle, spatula, etc. in 100 degree for overnight..

prepare 1% DEPC and keep for mixing in magnetic stirrer for overnight then autoclave it..

wipe out the place which you are going to use for RNA isolation with 70% ethanol and DEPC..

You can use the qiagen RNA extraction kit for RNA isolation.it gives you the high quality of RNA from tissue samples...

GOOD luck.

Korcan Ayata

Cleaning all equipment as suggested above is important.

Chopping and mixing may not be enough for a complete homogenization depending on the tissue.

A complete homogenization is required to make sure that your RNA extraction buffer/reagent penetrate all the tissue and inactivate RNases.

Eric R Hugo

I have replaced grinding in liquid nitrogen with homogenizing in tri-reagent (trizol) using a Qiagen Tissuelyzer (sp.?). The maching uses a stainless steel bead added to your sample to beat the tissue into solution. We use this method on adipose tissue and have pretty good success. After the trizol prep we further clean the RNA with a GE RNA miniprep kit (same as the Machery-Nagel kit). The process is somewhat tedious but we get very clean, intact RNA for both qPCR and cloning purposes.

Naveen Sathyan

I think Anil has a point here. Most of us take all precautions during RNA isolation but sometimes we overlook the electrophoresis part. I hope your electrophoresis unit is RNase free and you prepare your gel in RNase free buffer. Its always better to denature your RNA at 65 deg C for 5 min and placing on ice for 2 mins before loading your gel.

Not using Ice cold ethanol for washing might also lead to such results.

Good Luck.

Eman Hussein

If you use the same gel electrophoresis system for running DNA samples, you must clean it before use it to RNA , so I suggest to clean it with H2O2, and use new and autoclave tips, solutions also autoclave equipments to prevent any RNAse inhibitor.

Madhuram Khandelwal

Thanks to all for valuable suggestion........

Prakash Chandra

RNA Express reagent from HI-media is also giving good yields... I use it routinely for RNA Isolation... you just need to have the DEPC treated materials and A separate laminar hood ...

Selvaraj Milton Prabu

Try this protocol. But its not a revised one even best for neuroblastoma RNA isolation.

RNA Isolation with TRIzol (Invitrogen) and Qiagen RNAeasy

https://ocg.cancer.gov › files › RNA_Trizol_NBL

Shushruta Bhunia

Try to use dedicated glassware/chemical/pipettes/electrophoresis unit only for RNA. I did not use DEPC but fortunately I got intact RNA bands in gel with good conc. In my case high heat incubation of the glassware, mortal-pestle (after autoclaving) worked. And check the RNA once you isolate it as soon as possible. Good luck!!

Nikolay Klyashtornyy

To obtain intact RNA from tissue samples, it is crucial to follow proper protocols and precautions during the isolation process. Here are some suggestions to improve RNA integrity:

Proper tissue handling: Minimize RNA degradation by ensuring appropriate tissue handling techniques. Collect the tissue samples quickly after excision and keep them in RNase-free conditions. Use sterile instruments and wear gloves to avoid RNase contamination.

Quick homogenization: For tissue disruption, consider using a homogenization method that provides fast and efficient disruption while minimizing RNA degradation. Techniques such as mechanical homogenization, rotor-stator homogenization, or bead beating can be effective. Additionally, using liquid nitrogen during the homogenization step can help preserve RNA integrity.

RNase inhibitors: Incorporate RNase inhibitors into your extraction buffers to protect RNA from degradation. These inhibitors, such as RNaseOUT or SUPERase-In, can help preserve RNA integrity during the isolation process.

Gentle lysis and extraction: Avoid excessive vortexing or harsh lysis conditions that can lead to shearing and degradation of RNA. Use mild detergents or chaotropic agents, such as guanidine thiocyanate, to lyse the cells or tissues gently.

Precipitation and washing: Ensure proper precipitation and washing steps during RNA isolation. Use appropriate concentrations of ethanol or isopropanol, as well as the recommended centrifugation speeds and times, to obtain high-quality RNA.

DNAse treatment: Treat the isolated RNA with DNAse to remove any contaminating genomic DNA. This step helps ensure the purity of the RNA sample and prevents interference during downstream applications, such as cDNA synthesis.

Alternative methods: If you are experiencing challenges with the chopping and mixing method, you can explore other RNA isolation methods such as column-based RNA extraction kits (e.g., Qiagen RNeasy Mini Kit) or phenol-chloroform extraction methods. These methods often include specific buffers and protocols designed to optimize RNA yield and integrity.

RNA storage: Properly store the isolated RNA at -80°C or in liquid nitrogen to maintain its stability and integrity over time. Use RNase-free tubes and avoid repeated freeze-thaw cycles.

By implementing these precautions and considering alternative methods, you can improve RNA integrity and obtain intact RNA for downstream applications like cDNA synthesis and gene expression analysis.

These video playlists might be helpful to you:

https://www.youtube.com/playlist?list=PLzBiAPKi8vOPbE7Db9mKwloe34E8rXXEU

https://www.youtube.com/playlist?list=PLzBiAPKi8vONSqjcweFWHMRy13y3484bE

https://www.youtube.com/playlist?list=PLzBiAPKi8vOMheKwb0mu1dcZRaeGUFSlw

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