1.2 Sample Types

  1. What are the transport requirements for RNA and DNA samples?
    Overnight is a MUST for RNA for DNA it is preferred, avoid extreme temperatures. Surface mail is OK if it gets there in time.
  2. What are recommendations for whole blood extractions?
    Separate WBCs to avoid hemoglobin released upon hemolysis of RBCs. RBCs, when lysed release heme which is known PCR inhibitor.
  3. Three things to consider when transporting RNA or DNA samples?
    • -Shipping time
    • -Avoid extreme temperaturs
    • -If whole blood extraction separate WBCs
  4. Blood serum delivery requirements?
    • 1. separate from cells and freeze serum immediately to preserve RNA viruses
    • 2. 1-2ml is sufficient
    • 3. Serum should be shipped frozen on dry ice either DNA or RNA studies
  5. Plasma delivery requirements?
    • 1. Plasma samples are stable up to 5 days at 2-8 degrees C and longer if frozen at -20 to -70 degrees C
    • 2. Plasma should be shipped at 2-8degrees C and stored at -20 degrees C
    • 3. For RNA studies, extraction should begin within 4 hours, or specimen should be frozen. For long-term storage, serum, plasma, or blood cell nuclei can be prepared and stored at -20 to -70 degrees C.
  6. Bone marrow delivery requirements?
    • 1. Handle like whole blood, but usually get and need smaller samples
    • 2. Store at -20 degrees, stable for 2 weeks.
  7. Buccal cells delivery requirements?
    • No anticoagulant needed, because these are epithelial cells instead of blood cells.
    • 1. collect on cotton swabs, foam brush, or cytobrush
    • 2. store in closed container or Eppendorf-type tube
    • 3. ship at room temp or 4 degrees C (preferred)
    • 4. can use regular mail or overnight
  8. Cultured mammalian cells delivery requirements?
    • 1. Continuous incubation at 37 degrees C is recommended until cells are harvested for nucleic acid extraction.
    • 2. Fibroblasts, muscle cells, aminocytes, and tumor cells one T-25 flask
    • 3. For PCR 1 flask is needed; southern blot, more than 1 flask needed
    • 4. ship full of media, cells should be almost confluent in flask
    • 5. RNA testing possible ONLY if gene of interest is expressed in tissues culture version of the cell type
  9. Amniocytes/Amniotic fluid (prenatal specimens) expectation upon delivery requirements?
    • 1. Chorionic Villus sampling (CVS) specimens, cultured CVS cells, amniotic fluid, cultured cells from amniotic fluid, and any other cellular specimens derived from fetus prior to deliver.
    • 2. Maternal blood should accompany the specimen in order to carry out analysis
    • 3. A back up culture MUST be maintained until testing is complete
    • 4. Amniotic fluid can be processed without culture after 15 wks of gestation.
  10. Amniocytes/Amniotic fluid (prenatal specimens) delivery requirements?
    • 1. standard amount of amniotic fluid specimen is at least 10ml
    • 2. CVS specimen should be shipped in sterile tissue culture medium or saline buffer at Room Temperature
    • 3. Cultured cells from either amniotic fluid or CVS specimen should be shipped in two plastic culture flasks.
    • 4. DNA should be extracted from amniotic fluid specimen on the SAME day of arrival or the specimen should be store at 2-8 degrees C and the DNA should be extracted the NEXT day.
  11. Blood spots delivery requirements?
    • 1. Suitable for DNA analysis and are not recommended for studies requiring intact RNA.
    • 2. Protect from cross-contamination with other blood spots; dry well before shipping.
    • -reduces the chances of retained moisture encouraging growth of microorganisms
    • -also, each specimen should be separated by laboratory weighing paper, a paper coverslip, or by positioning them in such a way that they are not in contact with each other.
    • 3. stable for at least 19 months at 4-25 degrees C for PCR based testing (but need to remove hemoglobin for PCR)
  12. Hair bulbs delivery requirements?
    • 1. Used for forensic testing
    • 2. great for isolating DNA and doing PCR
  13. Chunks of tissue delivery requirements?
    • 1. SNAP FREEZE (quickly all the way through) for RNA or DNA 9-20 degrees C for at least 2 weeks; -70 degrees C for at least 2 years; 2-8 degrees C for up to 24 hours
    • -should be frozen because, solid tissue is a rich source of endogenous nucleases.
    • 2. for optimal results, tissue is snap frozen in liquid nitrogen.
    • 3. a tissue specimen should never be stored in a frost-free freezer, which makes freeze-thaw cycles, resulting in degradation of nucleic acids.
  14. Sputum, pus, urine delivery requirement
    • 1. Protect from cross contamination, refrigerate 2-8 degrees C
    • 2. Freeze if RNA based samples at -70 degrees C or lower, because ribonucleases continue to degrade RNA even at -20
    • 3.small amounts
    • 4. should be transported to labs at 4-8 degrees
  15. Extracted DNA samples delivery requirement
    • 1. Recommended that purified DNA samples be kept below the freezing point of water for long-term storage to minimize degradation activity of DNases
    • 2. Purified DNA can be stored safely in TE buffer at Room Temp for 26 weeks, at 2-8 degrees C for at lease one year if contaminating DNAases are absent, and for up to 7 years at -70 degrees C or lower.
    • 3. Transported at Room Temp and best as an Ethanol precipitate
  16. Paraffin-embedded tissue sample delivery requirements.
    • 1.  Difficult if crosslinker used or if old, but some protocols have been demonstrated to work at least some of the time.
    • -crosslinks in DNA occur when various exogenous or endogenous agents react with two different positions in the DNA
    • -DNA replication is blocked if the crosslink is not repaired. DNA polymerases cannot get past it.
    • 2. samples must be dewaxed with xylene or other agents and then rehydrated before nucleic acid isolation.
  17. Formalin-Fixed Paraffin Embedded Tissue delivery requirements.
    • 1. can be stored at room temp indefinitely for future DNA analysis
    • 2. not recommended for RNA studies
    • 3. If fixed in B5 are not  acceptable due to heavy metal interference with PCR reaction
    • 4. B5=HgCl2/37% Formalin (often used in Histology Labs)
    • 5. destroys lymphoid markers
    • 6. minimum of 2-5 micron sections
Author
Stephevette
ID
334948
Card Set
1.2 Sample Types
Description
clinical genetics
Updated