This protocol encourages the use of primary human tissues as a clinically relevant model for osteoarthritis research. Performing gene expression and histological analyses in these tissues may provide novel mechanistic insights. This technique standardizes methods for the identification and processing of human knee joint components.
It enables the extraction of high quality RNA that meets the requirements for downstream gene expression assays. It is challenging to obtain high quality RNA from diseased joint tissues, so the importance of seemingly insignificant steps, such as keeping everything clean and cool should not be underestimated. Demonstrating the procedure will be Thomas Wilson, a research associate and Navdeep Kaur, a postdoctoral fellow from my laboratory.
Begin by identifying the hard tissues, including cartilage, bone, and meniscus and soft tissues, including infrapatellar fat pad, anterior cruciate ligament, synovium and vastus medialis oblique muscle from the specimen based on the differences in the size, shape, color and texture. Cut each of the tissues into three sections. Rinse the tissue sections with sterile PBS to remove any residue or debris.
For RNA extraction, cut each of the three tissue sections into approximately one to two millimeter cubes. Transfer the smaller pieces to a two milliliter cryo vial. After securing the caps tightly, flash freeze the vials by submerging in liquid nitrogen for 30 seconds, then transfer the vial to the minus 80 degrees Celsius freezer for long-term storage, and repeat this procedure for all the tissues.
Homogenize the hard tissue using a mortar and pestle. Before homogenization, chill the mortar, pestle and spatula using liquid nitrogen to prevent the sample from thawing. Process only one sample at a time.
Transfer the tissue sample to mortar using a chilled spatula. Pour liquid nitrogen on top of the tissue. After the liquid nitrogen evaporates, crush the tissue with a pestle.
Repeatedly add liquid nitrogen while grinding the tissue to obtain fine tissue powder. Transfer the fine tissue powder in a 1.5 milliliter micro centrifuge tube pre-chilled by submerging in liquid nitrogen for 30 seconds. Add one milliliter of the ice-cold acid guanidinium phenol solution to each tube and incubate the tubes on ice for 20 minutes.
Before homogenizing different hard tissue, thoroughly clean the mortar, pestle and spatula with 70%ethanol, then with RNase decontaminant, followed by DEPC treated water. Soak the mortar, pestle and spatula in 70%ethanol, then wipe any residual liquid with a clean, lint-free tissue. Homogenize the soft tissue using the tissue homogenizer.
For disinfecting the homogenizer, wash the probe by running tubes of 70%ethanol and RNase decontaminant for 30 seconds per wash, then wash with DEPC treated water, followed by an additional 70%ethanol, each for 30 seconds. Wipe any residual liquid with a clean, lint-free tissue. Add one milliliter of acid guanidinium phenol solution to pre-labeled, five milliliter round bottom tubes, and transfer the tissue sample into the tube.
Place the tube on ice throughout the procedure and homogenize the tissue for a maximum of five 32nd pulses, or until the tissue is visually dissolved. Incubate the homogenized tissue on ice until all the tissue samples are processed. Disinfect and clean the homogenizer before processing the next tissue sample.
Using sterile forceps, remove any tissue chunk present in the teeth of the probe. Incubate all the homogenized tissue on the ice for additional 20 minutes and then transfer the tissue sample to a pre-labeled, pre-chilled 1.5 milliliter micro centrifuge tube. Seven unique human knee joint tissues obtained from osteoarthritis patients were processed within four hours of surgical removal.
The tissues were identified visually and confirmed by H&E histological staining. The H&E stained hard tissue sections, articular cartilage, subchondral bone and meniscus were visualized at six times magnification and 40 times magnification. Similarly, the H&E stained soft tissue sections, infrapatellar fat pad, anterior cruciate ligament, synovium and vastus medialis oblique muscle were visualized at six times magnification and then at 40 times magnification.
The quality assessment of extracted RNA indicated the presence of high quality samples based on integrity, yield and purity, as well as low quality samples, suggesting that despite using an optimized method, external factors like disease severity may impact the RNA quality across different tissues from the same patient. Correct identification of each tissue type is necessary to enable intra-and inter-tissue comparisons within and across patient samples, especially given the considerable heterogeneity of primary disease tissues. Sequencing technologies are rapidly evolving, and isolation of high quality RNA is a critical prerequisite to using these technologies to unravel underlying tissue-specific mechanisms in complex, chronic diseases like osteoarthritis.
