RNAlater™
Tissue preservation and RNA Stabilization Solution
• Aqueous, non-toxic tissue preservation
• Standardizes tissue preservation & nucleic acid isolation
• Provides spatial and temporal separation of collection and processing with no penalties in quality or throughput
• Samples in RNAlater can be stored:
at 37°C for one day
at 25°C for 1-2 weeks: ship samples at ambient temperature
at 4°C for months frozen indefinitely: archival storage of samples
Quality of RNA from RNAlater-treated Tissue
RNA Isolation: Sample Handling Summary
Process fresh samples as quickly as possible...
(some tissues may be more stable than others!)
.. or preserve in RNAlater and treat as fresh.
Still must be processed quickly into RNAlater,but can then be handled safely.
.. or snap-freeze and process frozen.
Must never thaw: however some small samples can be directly homogenized with a polytron.
Step 2 in RNA Isolation: Sample Disruption
Choice of disruption method critical for yield and quality
Dear Dr. Lader:
I am working at Johns Hopkins School of Medicine. Now we want to isolated RNA from mouse brain cortex for microarray. I have some problems for that and need your troubleshooting and some suggestion. I isolated total RNA following TRIZOL protocol, homogenizing tissue with Sonicator. After redissovling the RNA pellet with DEPC water, RNA concentration was round 2ug/ul. O.D. A260/280 was round 2.0.But when running the RNA at formaldehyde agarose gel (Northernax 10x MOPS gel running buffer, and Northernax formaldehyde load dye were from ambion a half year ago), the smear bands or nothing showed on the gel. I don't know what was wrong during isolation. Before I had worked another lab, my RNA quality was very good for RT-PCR, Northern, and RPA. Only different thing was using Polytron for homogenizing.
Your kind and help are greatly appreciated.
Best regards,
Step 3 in RNA Isolation:RNA recovery
Total RNA Isolation Methods
• Rapid
- one-step phenol-based - scalable
- glass-binding - higher potential throughput
• Phenol-free
- glass-binding
• Difficult tissues, specific challenges
- multi-step phenol based
The Hybrid RNA Isolation Method(for people who RNA isolation)
Requires: RNAqueous or equivalent
Acid Phenol:Chloroform
Protocol
• Disrupt sample in GiTC lysis buffer with Polytron
• Extract sample with 1:1 acid phenol:chloroform, spin and collect aqueous phase
• Continue with filter-based protocol
High yields
• Low residual protein (260:280 of ~2.0)
• Vacuum manifold adaptable
• Scalable 1mg - 1g tissue !!!
• Handles difficult tissues well
• No filter clogging problems
