Collecting Samples for RNA

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The protocol for collecting samples for RNA extraction

General tips

With next generation sequencing, it is important to consider all sources of possible contamination when collecting samples. It is also important to consider the study question. For example, best results for RNA extraction will come from flash-frozen tissue or tissue stored in RNA-later. It is important to consider the source tissue (because of tissue-specific expression) when designing an experiment with RNA.

A good resource is the RNA-seqlopedia and our RNA-seq page.

  • RNAases will “eat” DNA and they are everywhere. Use a sterilized surface. Sterilize surface with 10% bleach or UV light, then use RNAase Away to clean the surface.

  • RNA degrades in as little as 30 seconds, so tissues should be harvested quickly.

  • Use sterile blades and switch the blade between samples.

  • All centrifuge tubes used to store tissues should be sterilized and prepared before going out. In general, it is best practice to have tubes organized in a box, with at least 2 labels on each tube (in case one falls off). We have a special barcode label printer (with freezer-proof labels) that can be used.

Materials

  • Scalpels with replaceable, sterile blades

  • Autoclaved glass pyrex dish for cutting sample on and a flame to torch it; alternatively can use weigh boats that are RNAase/DNAase free

  • Autoclaved tweezers, probes, etc. Ideally use tweezers without the ridges, between which tissue gets stuck.

  • Ethanol in an autoclaved beaker for rinsing dissecting tools

  • A small flame for sterilizing dissecting tools between samples

  • Prepared, Autoclaved and labeled (with EtOH-proof pen) microcentrifuge tube(s)

  • RNA-ase away to wipe off surfaces and blades between samples

  • Nitex gloves

Tips for flash freezing in liquid N2

We use Fisher cryovials (#10-500-26), although note that they are really not designed to put into liquid N2. They work well for short-term storage in liquid N2 and then transfer to the -80C.

Tips for RNAlater

We have successfully placed tissue in RNAlater and flash frozen, with good results for RNA extraction.

If the tissue cannot be flash frozen in RNAlater, than it is important to:

(1) Get the tissue:RNAlater ratio correct following manufacturer’s directions: Trim the tissue to be less than 0.5 cm in at least one dimension and simply submerge it in 5 volumes of RNAlater solution (e.g., a 0.5 g sample requires about 2.5 mL of RNAlater solution).

(2) Keep the tissue at the correct temperature following the manufacturer’s direction for the specified amount of time before transferring to -80C. This allows the RNAlater to permeate the tissues. Do not freeze samples in RNAlater® Solution immediately; READ THE INSTRUCTIONS. Typically we store at 4°C overnight (to allow the solution to thoroughly penetrate the tissue), remove supernatant, then move to –20°C or –80°C for long-term storage. Although, we have had good luck flash-freezing tissues in RNAlater.

Samples can be stored in RNAlater at 4°C for one month, at 25°C for one week, or at –20°C indefinitely. Archive tissues treated with RNAlater solution at –20°C.

  • If any precipitation of RNAlater® Solution is seen, heat it to 37°C and agitate to redissolve it.

  • If the crystals do not go into solution at 37°C, loosen the cap and heat the solution at a higher temperature, up to 65°C, for ~30 minutes, mixing periodically. Once the crystals have dissolved, store the solution in smaller aliquots in case crystals re-form after cooling. Warming the solution in this way does not affect performance of RNAlater Solution.