RNA Extraction Protocol

Objective:Test quantity and quality of RNA extracted from fresh coral samples of 3 species (Montipora capitata, Porites compressa, and Pocillopora damicornis). This protocol includes a purification column which has not been used in the previous extractions and should increase quality.

Protocol: Hybrid Trizol (Invitrogen 15596018) and Direct-zol kit protocol (Zymo cat R2050)

Required Reagents and Expendables: Trizol (Invitrogen 15596018) 100% chloroform (molecular grade) 70% EtOH (molecular grade) RNAse free water (DEPC-treated water) RNA purification kit - Direct-zol™ RNA MiniPrep (Zymo Research Cat: R2050) RNase DNase free tubes with beads or garnets (MoBio 13123-50) bead beater or Tissuelyzer (Qiagen 85300) bench centrifuge at 4C (at least 12,000 x g) bench centrifuge at Room Temp (at least 12,000 x g) quick spin down centrifuge vortex Pipettes - P1000, P200, P10

Protocol Steps:

Complete these steps 1-12 in a hood

  1. add 1.0mL of Trizol in the 2mL garnet tubes

  2. bead beat 2mL garnet tubes with Trizol (Qiagen TissueLyzer frequency 1/s=30, time = 1min)

  3. incubate at RT for 5 min, hand mix several times

  4. spin down tubes using bench quick spin

  5. add 214µl of 100% chloroform and hand-shake for 15 sec

  6. incubate at room temperature for 3 min

  7. centrifuge at 12,000x g for 15 min at 4°C

  8. pipette out only the clear supernatant above the white interphase (volume should be ~500-600µl). add supernatant to a new RNase, DNase free 1.5mL tube

  9. add another 214µL of 100% chloroform to the garnet tube with sample and trizol and hand-shake for 15 sec

  10. incubate at room temperature for 3 min

  11. centrifuge at 12,000x g for 15 min at 4°C

    prepare your DNAse mix

  • for each reaction mix 5µl of DNase I (6U/µl) with 75µl of DNA Digestion Buffer (from Direct-zol kit R2050 Zymo)
  • Only mix enough for each day. Save on ice until use.

  1. pipette out clear supernatant (~200uL) into the RNase, DNase free 1.5mL tube from step 8 (DO NOT disrupt the interphase)

    Complete the following steps in the RNA prep area

  2. add 500uL of 70% Ethanol to the tube from steps 8 and 12, vortex for 3 sec and quick spin down

  3. pipette 500uL of RNA sample Ethanol mix onto the Direct-zol spin column

  4. centrifuge at 12,000x g for 15 sec at 4°C and discard the flow through

  5. pipette the remaining ~500uL of RNA sample and Ethanol mix onto the Direct-zol spin column

  6. centrifuge at 12,000x g for 15 sec at 4°C and discard the flow through

  7. add 400 μl Direct-zol RNA Wash Buffer to the column and centrifuge

  8. centrifuge at 12,000x g for 15 sec at 4°C and discard the flow through

  9. add 80µL of DNAse mix directly onto column and incubate at room temperature for 15 min

  10. Add 400 μl Direct-zol RNA PreWash to the column

  11. centrifuge at 12,000x g for 15 sec at 4°C and discard the flow through

  12. Add 400 μl Direct-zol RNA PreWash to the column

  13. centrifuge at 12,000x g for 15 sec at 4°C and discard the flow through

  14. transfer column onto new collection tube

  15. add 700µL of Direct-zol RNA Wash Buffer onto column

  16. centrifuge at 12,000x g for 2 minutes at 4°C and discard the flow through

  17. transfer column onto a new RNase, DNase free 1.5mL tube

  18. add 50µL of Direct-zol RNAse free water directly onto the column

  19. incubate at room temperature for 1 min

  20. centrifuge at 12,000x g for 2 minutes at 4°C and save the RNA

  21. aliquot 3µL of RNA sample in 200µl PCR tube for Nanodrop quantification

  22. aliquot 3µL of RNA sample in 200µl PCR tube for Bioanalyzer integrity check

  23. place RNA aliquot for nanodrop on ice and measure immediately.

  24. store RNA samples and bioanalyzer aliquots in -80C freezer

Samples were assessed on a ND-100

260/280 Description Nanodrop

“The ratio of absorbance at 260 nm and 280 nm is used to assess the purity of DNA and RNA. A ratio of ~1.8 is generally accepted as “pure” for DNA; a ratio of ~2.0 is generally accepted as “pure” for RNA. If the ratio is appreciably lower in either case, it may indicate the presence of protein, phenol or other contaminants that absorb strongly at or near 280 nm.”

260/230 Description Nanodrop “This ratio is used as a secondary measure of nucleic acid purity. The 260/230 values for “pure” nucleic acid are often higher than the respective 260/280 values. Expected 260/230 values are commonly in the range of 2.0-2.2. If the ratio is appreciably lower than expected, it may indicate the presence of contaminants which absorb at 230 nm.”

Results

Sample.Description Sample ng/µl A260 260/230 260/280
Blank RNase DNase free Water Blank 0.22 0.0005 0.20 5.77
Montipora capitata rep 1 MC_1 525.15 13.129 2.23 2.00
Montipora capitata rep 2 MC_2 525.33 13.133 2.21 2.00
Montipora capitata repeated column elution MC_E2 13.00 0.3255 1.59 1.80
Pocillopora damicornis rep 1 PD_1 449.81 11.245 2.37 2.07
Pocillopora damicornis rep 2 PD_2 446.80 11.17 2.32 2.07
Porites compressa rep1 PC_1 9.08 0.227 0.76 2.12
Porites compressa rep2 PC_2 9.03 0.226 0.78 1.84
Porites compressa repeated column elution PC_E2 5.09 0.127 0.97 1.66
Summary:

Montipora capitata and Pocillopora damicornis were extracted successfully. Porites compressa did not extract well. The majority of the RNA is being eluted in the first elution from the column. There is no need to do 2 elutions. This is a viable protocol for Montipora capitata and Pocillopora damicornis, but requires trouble shooting for Porites compressa.

Shinzato et al 2014 has demonstrated successful RNA extraction from Porites using a Qiagen kit. We have the Qiagen all prep kit in stock and will repeat the same species extractions as a comparison.

Shinzato C, Inoue M, Kusakabe M (2014) A Snapshot of a Coral “Holobiont”: A Transcriptome Assembly of the Scleractinian Coral, Porites, Captures a Wide Variety of Genes from Both the Host and Symbiotic Zooxanthellae. PLoS ONE 9(1): e85182. doi:10.1371/journal.pone.0085182

Written on May 3, 2016