One of the most problematic steps in RNA isolation is the first step - thorough lysis of the tissue or cell sample in a denaturant solution that inhibits RNA degradation by RNase. While it is possible to process fresh tissue directly, it is extremely important that all cells are disrupted immediately upon contact with the denaturant. This usually requires use of a polytron and even then some "difficult to process tissues" (e.g. hard tumors, bacterial cells, plant roots, etc.) are not effectively disrupted (see the article, "Cell Disruption: Getting the RNA Out "). Therefore, if you are having a problem with yield or degradation during RNA isolation, we usually recommend freezing the tissue sample before processing. Here we compare three methods for processing frozen tissues in a side-by-side test for quantity of mRNA recovered.
Freezing the Tissue
Samples should be frozen quickly so that the whole tissue sample freezes at once throughout. This may mean mincing the tissue into smaller fragments before freezing. Submerging the samples in liquid nitrogen will freeze the tissue pieces most quickly. Alternatively, a metal plate placed on dry ice can serve as a freezing surface.
Each of the methods below describes a distinct way of generating a tissue/cell lysate from which to purify RNA and is assessed for yield of poly(A+)RNA, when used to process 0.1 g of frozen mouse liver tissue. While the three methods each use a guanidine buffer to ultimately lyse the cells, they differ in how the tissue is processed prior or during that lysis step.
Method 1: Processing frozen tissue fragments in a dounce
Yield: 4.1 µg poly(A+)RNA
Frozen tissue is cut into small pieces (approx. 0.5 cm2) on dry ice, placed in a dounce, and processed as lysis buffer is added. Both pestle A and pestle B are used for ten strokes each.
Method 2: Processing frozen tissue fragments through a syringe
Yield: 3.2 µg poly(A+)RNA
Frozen tissue is cut into small pieces (approx. 0.5 cm2) on dry ice, added to lysis buffer and passed back and forth ten times through an 18 gauge syringe needle.
Method 3: Grinding the tissue to a powder in liquid N2
Yield: 7.1 µg poly(A+)RNA
The frozen sample is powdered by grinding the frozen tissue fragments in a prechilled mortar and occasionally adding liquid N2 into the mortar to prevent thawing. Once the tissue is ground to a fine powder, the denaturing solution is added to the mortar, and the semi-frozen mixture is stirred. This mixture can then be thawed and transferred to an appropriate vessel for further processing.
Note that by grinding the tissue to a powder in liquid N2 (Method 3), cellular disruption is much more complete resulting almost twice the yield of the other two methods.
