Mar 06, 2026
miRVana Total RNA (small and large RNA) Isolation Procedure of Human Knee Joint Tissues
- Priya Kulkarni1,
- Glyn D Palmer2,
- Kyle D Allen1
- 1Department of Biomedical Engineering, University of Florida, Gainesville, Florida 32611, United States;
- 2Department of Orthopaedic Surgery and Sports Medicine, College of Medicine, University of Florida, Gainesville, Florida 32611, United States
- University of Florida
Protocol Citation: Priya Kulkarni, Glyn D Palmer, Kyle D Allen 2026. miRVana Total RNA (small and large RNA) Isolation Procedure of Human Knee Joint Tissues. protocols.io https://dx.doi.org/10.17504/protocols.io.n92ldx7mog5b/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: March 06, 2026
Last Modified: March 06, 2026
Protocol Integer ID: 269159
Keywords: mirvana total rna, isolation procedure of human knee joint tissue, human knee joint tissue, best quality of rna, rna, joint tissues from frozen clinical biopsy, large rna, different types of knee, knee, joint tissue, frozen clinical biopsy
Funders Acknowledgements:
Priya Kulkarni
Grant ID: UC2 AR082196
Glyn D Palmer
Kyle D Allen
Grant ID: UC2 AR082196
Abstract
This protocol enables the user to extract best quality of RNA from different types of knee joint tissues from frozen clinical biopsies.
Guidelines
'Tissue harvest' described under this protocol requires prior approval by the users' Institutional Ethics Board or equivalent ethics committee
Materials
RNase Away and 70% Ethanol (if needed) spray bottle to clean tools and workspace
Liquid Nitrogen (LN2)
Dewar for Liquid Nitrogen
Wet ice bucket
Frozen beads / Dry Ice to keep samples frozen
Autoclaved paper foil
Autoclaved tool set (for tissue homogenization) – scalpel for tissue cutting, straight artery forceps for tissue handling, stainless steel hammer for tissue smashing / dissociation
BeadBug Microtube homogenizer (Benchmark Scientific, Cat No #D1030)
Bead/Bug homogenizer microtubes (2ml) (Benchmark Scientific, Cat No #D1033)
Kim Wipes and paper towels for tissue removal, handling and cleaning
1.5 ml / 2ml sterile, RNase free Eppendorf tubes for organic isolation and collection
Precooled 4°c benchtop centrifuge
100% Ethanol (for the precipitation and concentration of RNA from aqueous solutions)
RNAlater™-ICE Frozen Tissue Transition Solution (Invitrogen, Cat No. #4427575)
mirVana™ miRNA Isolation Kit, without phenol (Invitrogen, Cat No. #AM1561) including Lysis Buffer, miRNA Homogenate additive, wash solutions, elution solutions, columns and collection tubes
Acid-Phenol:Chloroform, pH 4.5 (with IAA, 125:24:1) (Invitrogen, Cat No. #AM9720)
Glycogen (Invitrogen, stock 5 mg/ml) for improved RNA precipitation
Troubleshooting
A. Tissue Harvest
Harvest joint human knee joint tissues including cartilage/bone, synovium and infra-patellar fat-pad (IFP) immediately following surgery and flash freeze in LN2. (Note: Dissected tissues should be ≤ 0.5 cm in all dimensions.)_
The following day add 10 volumes of RNAlater™-ICE to each frozen tissue sample and store at -20 °C or colder for at least 16 hours. (Note: Typical tissue weights are 100 – 300 mg.)_
B. Tissue Homogenization
Remove tissue pieces from RNAlater™-ICE and transfer samples to autoclaved foil. (Note: Samples should have a uniform blue color indicating penetrance of the RNAlater™-ICE throughout the entire tissue.)_
Using a hammer or pestle and mortar, pulverize the tissue into small pieces. (Note: Homogenization may be performed in the presence of LN2 to facilitate grinding the tissue into a fine powder. ‘Elastic’ tissues such as meniscus and cartilage can also be finely minced using a scalpel to further aid homogenization.)_
Transfer samples to sterile, RNase-free Eppendorf tubes to perform tissue lysis and mRNA isolation according to the mirVana™miRNA Isolation Kit reagents and protocol.
A. Tissue Lysis
Add Lysis/Binding buffer (10 volumes per tissue mass, ~1-2 ml) to Bead/Bug homogenizer microtubes.
Transfer the dissociated tissue into Bead/Bug homogenizer microtubes and further homogenize it at maximum speed, for 3 mins. After homogenization transfer tubes to ice for 2 mins and repeat the cycle an additional 3 times (Note: Total tissue homogenization time could be extended up to 4 mins)._
Spin the tubes at 12,000 rpm for 5 minutes to remove tissue debris.
Transfer a clear tissue lysate to sterile/RNase-free 1.5 ml/2ml Eppendorf tubes. Avoid tissue fragments. Fat pad samples will contain a thin layer of fat at the surface which should also be avoided.
B. Organic Extraction
Add 1/10 volume of miRNA Homogenate Additive to the tissue lysate (e.g. 100 ul for 1 ml). Mix well by vortexing or inverting.
Leave the samples on ice for 10 mins.
Add Acid-Phenol:Chloroform at a volume equal to the lysate prior to addition of miRNA Homogenate Additive and vortex 30 sec to mix.
Centrifuge for 15 min at 12,000 rpm at 4°C to separate aqueous and organic phase. After centrifugation the interphase should be compact. If not repeat centrifugation.
Carefully remove the aqueous (upper) phase without disturbing the lower phase and transfer to a fresh sterile/RNase-free 1.5 ml/2ml Eppendorf tube. Be conservative, do not touch the interface. Note the final volume of the aqueous phase of each sample.
Add 1-2 uL glycogen (Invitrogen, stock 5 mg/mL; final concentration of 50-100 ug/mL) to aid RNA precipitation and mix.
Add 1.25 volumes of RT 100% ethanol to the aqueous phase and mix by vortexing.
C. Total RNA Isolation Procedure
Preheat Elution solution to 95°C
Prepare Filter Cartridge and collection tube for each sample.
Transfer lysate/ethanol mixture into Filter cartridge. Up to 700 ul can be applied to each cartridge and centrifuge for ~30 sec at 10,000 rpm.
Discard flow through and repeat step with remaining lysate/ethanol mixture, until all the sample has been spun through.
Wash each cartridge by applying 700 ul miRNA Wash Solution 1. Centrifuge for ~30 sec at 10,000 rpm). Discard flow through and re-use the same collection tube.
Apply 500 ul Wash Solution 2/3 and repeat centrifugation.
Discard flow through and spin again using the same collection tube at 15,000 rpm for 2 min, to remove any residual fluid from the filter.
Apply 80 ul of preheated Elution Solution to the center of the filter. Touch the center of the filter gently with the pipette tip prior to dispensing to ensure the liquid is absorbed by the filter. Avoid getting Elution Solution on the sides of the Filter Cartridge. Close the cap and spin at 12,000 rpm for 1 min at maximum speed to recover RNA.
Collect the eluate and pass through the filter again with an additional spin at 12,000 rpm for 1 min to maximize mRNA yield from the column. (Note: Can reduce the volume of elution buffer up to 70 ul, to increase the concentration of RNA in the elution step)_
Use Nanodrop for Spectrophotometer readings.
Target specifications: RNA Concentration: 40 – 100 ng/ul, A260/280 ratio: 1.8 – 2, A260/230: 1.6, RIN value: >6
Acknowledgements
This protocol was developed under 'REJOIN Transcriptome study', a part of NIH funded consortium (UC2 AR082196). All the authors thank NIH for their help in execution of this study.