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DNA/RNA Extraction of Pentagona and Hystera Sea Stars

DNA/RNA Extractions on 8 Sea Star Tissue Samples Stored in DNA/RNA Shield

Notes

Sample Preparation

  • Samples:
    • CPAB-003
    • CHSB-005
    • CPBO-008
    • CPOI-009
    • CPAB-011
    • CPDB-001
    • CPBP-001
    • CHSY-010
  • Thawed samples on ice bucket
  • Prepared forceps, foil, and razor blades
  • Cleaned forceps with 10% bleach, DI water, 70% ethanol, and RNaseZap before and between each sample
  • Made 1 bead tube per sample with 700ul of DNA/RNA shield in each
  • Cut each tissue piece in half and placed in the bead tube
  • CPAB-003 1
  • CHSB-005 2
  • CPBO-008 3
  • CPOI-009 4
  • CPAB1-011 5
  • CPDB-001 6
  • CPBP-001 7
  • CHSY-010 8
  • Vortexed the bead tubes for 1 minute at max speed
  • The tissue didn’t look that broken up but I continued with the protocol that I had tired previously
  • Spun down the tubes to remove bubbles (there were still some)
  • Removed all the volume I could from the bead tubes into new 1.5mL tubes ~450ul
  • Saved the bead tubes by adding 300ul DNA/RNA Shield to each and freezing them. They looked like: 9

DNA Extraction

  • Added 22.5ul Proteinase K and 45ul Pro K digestion buffer to each 1.5mL tube
  • Vortexed and spun down the tubes
  • Added equal volume (520ul) of DNA/RNA lysis buffer to each tube
  • Flicked and inverted tubes to mix and spun down
  • Warmed 10mM Tris HCl pH 8 and nuclease free water in the thermomixer at 70 degrees C
  • Added 700ul of the liquid to yellow spin columns and collection tubes
  • Centrifuged 16,000rcf for 30 seconds
  • Saved the flow through in new 5mL tubes
  • Added the remaining liquid to the yellow spin columns
  • Centrifuged 16,000rcf for 30 seconds
  • Saved the flow through in the 5mL tubes
  • Added 400ul DNA/RNA prep buffer to the yellow spin columns
  • Centrifuged 16,000rcf for 30 seconds
  • Discarded flow through (Zymo kit waste)
  • Added 700ul DNA/RNA wash buffer to the yellow spin columns
  • Centrifuged 16,000rcf for 30 seconds
  • Discarded flow through (Zymo kit waste)
  • Added 400ul DNA/RNA wash buffer to the yellow spin columns
  • Centrifuged 16,000rcf for 2 minutes
  • Transferred spin columns to new 1.5mL tubes for each sample labeled as final DNA tubes
  • Discarded flow through and collection tubes
  • Added 50ul warmed 10mM Tris HCl to the spin columns gently by dripping over the filter
  • Incubated at room temp for 5 minutes
  • Centrifuged 16,000rcf for 30 seconds
  • Added another 50ul warmed 10mM Tris HCl to the spin columns gently by dripping over the filter
  • Incubated at room temp for 5 minutes
  • Centrifuged 16,000rcf for 30 seconds
  • Discarded the spin columns
  • Made strip tubes with 7ul of each sample in them for QC
  • Kept tubes on ice bucket then stored in -20 degree freezer

RNA Extraction

  • Added equal volume (1000ul) 100% ethanol to each 5mL tube
  • Vortexed and spun down
  • Added 700ul of the liquid to green spin columns and collection tubes
  • Centrifuged 16,000rcf for 30 seconds
  • Discarded flow through (Zymo kit waste)
  • Repeated addition for the remaining liquid (2 times) to the green spin columns
  • Centrifuged 16,000rcf for 30 seconds
  • Discarded flow through (Zymo kit waste)
  • Added 400ul DNA/RNA wash buffer to the green spin columns
  • Centrifuged 16,000rcf for 30 seconds
  • Discarded flow through (Zymo kit waste)
  • Created the DNase mix:
    • 75ul DNA digestion buffer * 8 = 600ul
    • 5ul DNase I * 8 = 40ul
  • Flicked and spun down mix
  • Added 80ul DNAse mix to each green spin column filter
  • Incubated for 15 minutes at room temp
  • Added 400ul DNA/RNA prep buffer to the green spin columns
  • Centrifuged 16,000rcf for 30 seconds
  • Discarded flow through (Zymo kit waste)
  • Added 700ul DNA/RNA wash buffer to the green spin columns
  • Centrifuged 16,000rcf for 30 seconds
  • Discarded flow through (Zymo kit waste)
  • Added 400ul DNA/RNA wash buffer to the green spin columns
  • Centrifuged 16,000rcf for 2 minutes
  • Transferred spin columns to new 1.5mL tubes for each sample labeled as final RNA tubes
  • Discarded flow through and collection tubes
  • Added 50ul warmed nuclease free water to the spin columns gently by dripping over the filter
  • Incubated at room temp for 5 minutes
  • Centrifuged 16,000rcf for 30 seconds
  • Added another 50ul warmed nuclease free water to the spin columns gently by dripping over the filter
  • Incubated at room temp for 5 minutes
  • Centrifuged 16,000rcf for 30 seconds
  • Discarded the spin columns
  • Made strip tubes with 5ul of each sample in them for QC
  • Kept tubes on ice bucket then stored in -80 degree freezer

QC

Broad Range Qubit for DNA and RNA

Sample Reading 1 (ng/ul) Reading 2 (ng/ul) Average DNA (ng/ul)
Standard 1 190 RFU - -
Standard 2 21286 RFU - -
CPAB-003 62.4 61.4 61.9
CHSB-005 7.18 6.96 7.07
CPBO-008 4.28 4.2 4.24
CPOI-0009 10.7 10.6 10.65
CPAB1-011 19.9 19.7 19.8
CPDB-001 17.5 17.4 17.45
CPBP-001 18.6 18.5 18.55
CHSY-010 12 11.9 11.95
  • RNA
Sample Reading 1 (ng/ul) Reading 2 (ng/ul) Average RNA (ng/ul)
Standard 1 408 RFU - -
Standard 2 8288 RFU - -
CPAB-003 25.2 24.8 25
CHSB-005 too low - -
CHSB-005 too low - -
CPBO-008 too low - -
CPOI-0009 too low - -
CPAB1-011 too low - -
CPDB-001 too low - -
CPBP-001 13.4 12.8 13.1
CHSY-010 too low - -

1% Agarose Gel for DNA Quality

RNA TapeStation

Notes

  • Quality of DNA and RNA is good but yield is LOW especially of the RNA
  • I think the homogenization was not efficient and also the small sample pieces need the most efficient
  • Next extraction I will try to chop the pieces before homogenization and maybe add incubation too
Written on October 15, 2020