I grind the heart up first with a mortar and pestle under liquid nitrogen, then scoop some of the powder/chunks into trizol, and some into several different lysis buffers for various protein applications using a metal spatula also dipped in liquid nitrogen. As soon as the powder/chunks are in the trizol or lysis buffer, I either further homogenize with our electric homogenizer,, or qucik freeze the tube in liquid nitrogen for later. Usually I work on protein first and freeze the trizol for later.

Our lab has the Bullet Blender homogenizer from Next Advance. You can purchase various bead sizes depending on the sample to be homogenized. It works very well and you can homogenize up to 24 samples at one time. Highly recommended for any type of tissue homogenization!

I grind the heart up first with a mortar and pestle under liquid nitrogen, then scoop some of the powder/chunks into trizol, and some into several different lysis buffers for various protein applications using a metal spatula also dipped in liquid nitrogen. As soon as the powder/chunks are in the trizol or lysis buffer, I either further homogenize with our electric homogenizer,, or qucik freeze the tube in liquid nitrogen for later. Usually I work on protein first and freeze the trizol for later.

I think better to use kit and just follow the instruction of manufacturer company. No need to use liquid nitrogen, homogenizer and trizol. Trizol also is very carcinogen and you must be very careful when you use it. TQ

Muscle lysis is challenging due to the presence of the muscle fibers. You need a mechanical method such as mortar/liquid nitrogen or ceramic bead homogenizer (I use the one from Roche but others are good too). For the buffers, I follow Chomczynski and Sacchi method using guanidinium for the lysis (in association with the beads) and phenol chloroform extraction for the recovery of RNA.

It works well.

If you are doing multiple sample, liquid nitrogen is not ideal since you do not want to carry over any material from one prep to the other unless you have many mortars and pestles. A bead homogeniser is ideal if you have one. I had a similar issue with extracting RNA from gut. We did not have either a homogeniser or a bead beater (ceramic beads). I ended up putting tissue in 500 ul Trisol in an eppendorf tube, adding about 30-50 ul of a well washed slurry of silicon carbide (medium mesh), and grinding the tissue with a disposable plastic pestle (sigma). Not elegant but it seemed to work. Silicon carbide is the black material you find on sandpaper / nail files.

Ian Teo

Been using several tools for homogenization and in my new lab I could have the luxury of choosing which method I prefer! After using the Tissue Lyser of Qiagen I will never use anything else, in less than 5 min you get all your samples done (48 per time). And it works so good with any type of tissues... In one load you can get so much done and so fast... (we got ours on trial use, you can get in contact with some sale representatives and ask them to borrow a machine for a while...). This is the most time efficient method I ever tried!

The only problem is that the shaking may cause some heat but you can reduced it just by pre-freezing the shaking blocks on dry ice/ or in -80 and by keeping the machine in a cold room.

Bullet blender and all the other methods have been abandoned since its arrival!

Trizol and any standard rotor-stator tissue homogenizer work well for muscle samples.

I find using kits designed for extraction of RNA from fibrous tissues very helpful. It basically adds a Proteinase K incubation step to the standard protocol and this results in a higher RNA yield.

Thank you all,

I am going to use a polytron style homogenizer for the initial steps.

Thanks

Michael

You can please follow the links below to obtain certain crucial informations:-

1. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3706331/

2. https://lifescience.roche.com/wcsstore/RASCatalogAssetStore/Articles/BIOCHEMICA_1_06_p9-10.pdf

You can also download pdf of the file attached herewith.

For physical grinding, use a bead dispenser to load your vials and plates with your desired amount of beads.

Here is a bead dispenser that I work with. Also using a large number of samples. LabTIE also has a large variety of Bead dispensers, fitting tubes, vials, 96-well 384-well etc.

https://www.labtie.com/seed-bead-powder-dispensers/112346-bead-dispensers

https://www.labtie.com/seed-bead-powder-dispensers/112346-bead-dispensers