TABLE 6

Sample Processing and Storage Tips for DNA/RNA

ParameterDNARNA
Source• The best quality and quantity of material are extracted from fresh or frozen solid tissue, followed by (in decreasing order) tissue preserved but not fixed, tissue fixed shortly after collection209 but not embedded, and tissue fixed and quickly embedded225• RNA should be extracted from fresh or snap frozen material at –70°C or below225,226
• DNA can be obtained from sections stained with hematoxylin and eosin,225 but toluidine blue is preferred if microdissection is required225• RNA can be extracted from fixed embedded tissue, but it has usually suffered more from the treatment of the specimen before fixation than from the processing itself225
• When DNA is isolated by LCM from frozen solid tissue, relative humidity in the laboratory must be controlled231• When LCM is used, and to avoid RNA degradation, it is imperative to keep tissue sections as dry as possible after fixation231
• Maintain tissue in desiccators after staining and during transport231
• Maintain relative humidity near 20% during LCM231
Time to freeze• Freeze immediately after collection• Freeze immediately after collection
• Solid tissues should not remain more than 30 min after death or surgery removal without optimal storage conditions to prevent degradation18,210,229• Solid tissues should not remain more than 30 min after death or surgery removal without optimal storage conditions to prevent degradation18,210,229
Extraction protocol• DNA can be extracted from frozen or formalin-fixed and paraffin-embedded tissues reliably with QIAmp Micro Qiagen (Qiagen Inc, Valencia, CA)210• TRIzol reagent (Thermo Fisher Scientific, Waltham, MA) or traditional phenol-chloroform extraction methods279 are often used for RNA isolation from fresh or frozen samples56,210
• Several other methods have been also tested in postmortem tissue derived from brain, spleen, liver, heart, intestine, and oral tissue101,280282• RNeasy Mini Kit (Qiagen Inc) provides RIN values much higher that TRIzol reagent177
Tissue fixation and preservation• If fixation is needed, 4% buffered formalin is less DNA damaging than the 10% nonbuffered formalin.229 The shorter the time a tissue is stored in buffered formalin fixation, the better the DNA preservation229• Immediate immersion of tissue sample in TRIzol reagent is currently used in many laboratories to improve RNA preservation210
• Periodical renewal of the buffered fixative solution reduces the deleterious effects of old formalin210• Hepes-Glutamic Acid Buffer-Mediated Organic Solvent Protection Effect fixation can be also successfully used283
Storage• DNA can be successfully preserved for several years if stored at –80°C or below,24,184,210 whereas some suggest that DNA is stable at 4°C for at least 3 y284• RNA can be successfully preserved if stored at –80°C or below24,184
• For optimal preservation, formalin-fixed, paraffin-embedded tissue should be stored as a block. Paraffin blocks should be stored at temperatures below 27°C in an area with pest and humidity control24• For optimal preservation, formalin-fixed, paraffin-embedded tissue should be stored as a block. Paraffin blocks should be stored at temperatures below 27°C in an area with pest and humidity controls24
Freeze/thaw steps• Unnecessary thawing and refreezing of frozen biospecimens or frozen samples of biomolecules extracted from the biospecimens should be avoided24• RNA integrity is severely affected by freeze/thaw steps24,233
Detection technique• PCR is commonly used to detect genomic or viral DNA from fresh tissue, or from LCM, formalin-fixed, and paraffin-embedded postmortem tissues67,282,285,286• RNA quality can be detected with high reproducibility and sensitivity with the microfluidic chip-based capillary electrophoresis270,271 using a Bioanalyzer (Agilent Technologies, Santa Clara, CA) or Experion (Bio-Rad Laboratories, Munich, Germany) instrument
  • LCM, laser microdissection.