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    Test Scallop RNA Extractions
    • Notes
    • Metadata
    • Relevant Items
    • RNA Extraction - Trizol and PureLink Kit
    • purelink_rna_mini_kit_man.pdf
    Editing disabled on read-only entry.

     
    Test Scallop RNA Extractions

    Monday, 6/5/2017On column DNase treatmentTuesday, 6/6/2017Table1170607-Pfstr-He_Eukaryote Total RNA Nano_DE13806560_2017-06-07_10-46-38.pdfWednesday, 6/7/2017Table2Tuesday, 7/11/2017Table3Monday, 7/17/2017Pfister_Total_RNA_Nano_2017-07-17_1of1_AC.pdfTable4Wednesday, 7/26/2017Thursday, 8/24/2017EZNA testMonday, 8/28/2017Tuesday, 9/12/2017EZNA vs PureLink with Liquid Nitrogen
    Monday, 6/5/2017
    Extracting RNA from rock scallop tissues for transcriptome assembly. Today will just be doing 8 samples as I am still pretty new to RNA extraction.
    Samples
    358 - WA (Dabob)
    65 - WA (Seiku/outer coast)
    1.
    358_ctenidia
    2.
    358_mantle
    3.
    358_adductor
    4.
    358_gonad
    5.
    65_ctenidia
    6.
    65_mantle
    7.
    65_adductor
    8.
    65_gonad
    Prepared fresh DNase I treatment (Quaigen) by adding RNAse free water to lyophilized DNAse.
    750 uL Trizol, 150 uL chloroform
    Tuesday, 6/6/2017
    Quantifying with Qubit (RNA Broad Range) and Nanodrop. Also dropped off 10 uL aliquots to the Functional Genomics core for a Bioanalyzer run.
    260/280 = 1.8-2.2
    260/230 > 1.7
    ●
    I think something funky happened with sample 358_gonad, either with the Nanodrop or Qubit so will likely redo.
    ●
    The ctenidia did best and the adductor did worst, both with weird 260/230 values and low concentration. I need to figure out a better way to mix up adductor muscle tissue.
    Bioanalyzer results
    ●
    Most had RIN values over 9 (which is good)
    ●
    358_gonad had a weird peak that was the same even with a rerun Bioanalyzer. Possibly DNA contamination?
    ●
    358-M: RIN 6.9, showing signs of degradation
    ●
    65-G: RIN 6.6 - some degradation
    170607-Pfstr-He_Eukaryote Total RNA Nano_DE13806560_2017-06-07_10-46-38.pdf
    Tips on reading RNA bioanalyzer results here.
    Wednesday, 6/7/2017
    Trying out some modifications to the protocol. In particular, using a pestle rotor motor for grinding the tissue, and working faster through steps. I also messed up the on column DNase treatment a little so trying to improve that.
    358 - WA (Dabob)
    248 - WA (Dabob)
    302 - AK
    380 - CA
    1.
    358_Abductor_B
    2.
    248_gonad
    3.
    248_mantle
    4.
    65_abductor_B
    5.
    65_gonad_B
    6.
    302_ctenidia
    7.
    C380_ctenidia
    8.
    302_mantle
    Submitting to the Functional Genomics Core for Qubit and Bioanalyzer
    Tuesday, 7/11/2017
    Abductor: 1 WA Seiku?, maybe 1 WA dab
    Ctenidia: 1 WA Seiku, 1 WA Dab, maybe 1 AK and 1 CA
    Mantle: 1 WA Sekiu, maybe 1 WA Dab and 1 AK
    Gonad: maybe 1 WA Dab and 1 WA Sekiu
    1.
    302_Abductor (AK)
    2.
    380_Abductor (CA)
    3.
    380 Mantle (CA)
    4.
    380_Gonad (CA)
    5.
    302_Gonad (AK)
    6.
    248_Abductor (WA Dab)
    7.
    248_Ctenidia (WA Dab)
    8.
    56_Ctenidia (WA Sekiu)
    Monday, 7/17/2017
    Bioanalyzer QC report
    Pfister_Total_RNA_Nano_2017-07-17_1of1_AC.pdf
    Day 0 Quality RNA
    Wednesday, 7/26/2017
    RNA Extractions from Day 1:
    Ambient
    ●
    AK: 305, 311
    ●
    WA_Sekiu: 241, 53
    ●
    WA_Dabob: 360, 359
    ●
    CA: 391, 390
    Low pH
    ●
    AK: 310, 312
    ●
    WA_Sekiu: 36,39
    ●
    WA_Dabob: 361,242
    ●
    CA: 372,370
    Day 2 Low pH
    ●
    WA_Sekiu: 66,51
    ●
    WA_Dabob: 240, 236
    ●
    CA: 373, 374
    Plan is to do 12 libraries - 4 from Day 1 ambient, 4 from Day 1 low pH, and 4 from Day 2 Low pH
    Thursday, 8/24/2017
    Do 8 extractions with EZNA RNA Mollusc kit to see if it works better for tricky tissues like abductor and gonad.
    ●
    36_ ctenidia: 350uL, thick milky layer, took out most liquid for pellet
    ●
    310_ctenidia: 350uL, thick milky layer
    ●
    310_abductor: 400uL, lots of milky layer, took out most liquid
    ●
    361_abductor: 400uL, serrated pestle, lots of milky
    ●
    370_gonad: 350uL, brown tissue, serrated pestle, thick milky, took out most liquid
    ●
    36_gonad: 400uL, orangy, almost no milky
    ●
    370_mantle: 400uL, not much milky
    ●
    361_mantle: 400uL, not much milky, took out most liquid
    Only really observed a pellet in 1 tube.
    Monday, 8/28/2017
    EZNA with liquid nitrogen
    ●
    312_abductor: LN+pestle, lots of milky, no pellet
    ●
    39_abductor: LN+pesrle, some white in milky interface, no pellet
    ●
    312_mantle: LN+pestle (not well homogenized), little milky, pellet
    ●
    242_mantle: LN+mortar, some milky, pellet
    ●
    372_gonad: LN+mortar, lots of milky, pellet
    ●
    242_gonad: LN+pestle, some milky, pellet
    ●
    370_ctenidia: LN+pestle, lots milky, no pellet
    ●
    39_ctenidia: pestle, some milky, pellet
    Tuesday, 9/12/2017
    Trizol PureLink extraction with liquid nitrogen. 100ul elution run through 2x
    ●
    312_abductor
    ●
    39_abductor
    ●
    312_mantle
    ●
    242_mantle
    ●
    372_gonad
    ●
    242_gonad
    ●
    370_ctenidia
    ●
    39_ctenidia
    Clearly the PuereLink kit is more reliable and has greater yield. Will use that going forward.

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