BioMasher™

RNA Isolation Using The BioMasher

The BioMasher™ Sample Preparation Device can be used on variety of samples to create a whole cell homogenate that will serve as the starting point for a variety of RNA extraction kits and protocols.

We recommend homogenizing the sample in the presence of a Guanidine-based lysis buffer (such as RNA Lysis Solution of the Cartagen Total RNA Isolation Kit or that supplied by most commercially available RNA isolation kits) to inactivate the RNases during homogenization. Optionally, the BioMasher may be pre-treated by either of the following methods:

Optional Method 1: Rinse BioMasher Homogenizer, Column Insert, and Collection Tube with a solution of 0.1 M NaOH and 1 mM EDTA followed by RNase-free water.

Optional Method 2: Rinse BioMasher Homogenizer, Column Insert, and Collection Tube with chloroform.

The following is an example of how to incorporate the BioMasher into the Cartagen Total RNA Isolation Kit and the Qiagen RNeasy Mini Kit with plant samples. Please review the Qiagen manual for advice on normal precautions to take when working with RNA.

Cartagen Total RNA Isolation Kit
At Step 2 of the Cartagen Total RNA Isolation Kit protocol:
For each sample to be processed, place the desired quantity of plant tissue into the BioMasher column filter insert, add 50 µL of RNA Lysis Solution, homogenize the sample with the BioMasher (either by hand or attached to a lightweight handheld power drill). Remove the homogenizer and add an aditional 150 µL of RNA Lysis Buffer. Replace the homogenizer and spin in a microcentrifuge for 30 seconds at 10,000 rpm. Remove and dispose of the column insert and the homogenizer. Add the necessary amount of RNA Lysis Solution to the sample to equal the final volume for the sample described in Step 2, and proceed with Step 4 of the protocol (add 0.5 volume of 100% ethanol…).

Qiagen® RNeasy® Plant Mini Kit
Please follow the notes in the Qiagen protocol to prepare the lysis buffer to be used. The RNeasy Kit provides two different lysis buffers: RLT (Guanidine Isothiocyanate) and RLC (Guanidine Hydrochloride). ß-Mercaptoethanol must be added to either the RLT or RLC buffer prior to use.
At Step 2 of the Qiagen RNeasy Mini Protocol for Isolation of Total RNA from Plant Cells and Tissues and Filamentous Fungi (page 75 of the RNeasy Mini Handbook 06/2001):
For each sample to be processed, place up to 50 mg (do not exceed 100 mg as per the instructions) of fresh or frozen plant tissue into the BioMasher column insert, add 50 µL of Buffer RLT or Buffer RCL, homogenize the sample with the BioMasher (either by hand or attached to a lightweight handheld power drill). Remove the homogenizer and add an additional 150 µL of Buffer RLT or Buffer RLC. Replace the homogenizer and spin in a microcentrifuge for 30 seconds at 10,000 rpm. Remove and dispose of the column insert and the homogenizer. Add 250 µL of Buffer RLT or Buffer RLC to the microcentrifuge tube containing the cell lysate and proceed with Step 4 of the protocol (pipet the lysate directly onto a QIAshredder spin column…).

Other Kits and Methods
Other kits and methods can easily be adapted for use with the BioMasher. Simply add 50 µL of the initial Guanidine containing lysis buffer along with the required amount of plant tissue (usually 50-100 mg) into the BioMasher column insert. Homogenize the sample with the BioMasher (either by hand or attached to a lightweight handheld power drill). Remove the homogenizer and add an additional 150 µL lysis buffer. Replace the homogenizer and spin in a microcentrifuge for 30 seconds at 10,000 rpm. Remove and dispose of the column insert and the homogenizer. The total amount of lysis buffer at this point is approximately 200 µL. If the kit or method normally starts off with a larger amount, adjust the volume by adding additional lysis buffer to the microcentrifuge tube containing the cell lysate so that the final volume equals the amount used in the kit or method. Then continue with the next step in the protocol.

Trademarks:

Cartagen is a trademark of Cartagen Molecular Systems Inc.

RNeasy, QIAshredder, and Qiagen are trademarks of Qiagen GmbH.