From fecf8e8186e45ae26c2dca753e492377d5f56184 Mon Sep 17 00:00:00 2001 From: Sarah Tanja Date: Tue, 22 Aug 2023 17:59:15 -0700 Subject: [PATCH] rna woes trying to make sense of phenol --- .../rna-woes/index/execute-results/html.json | 14 + docs/index.html | 58 +- docs/listings.json | 4 +- .../PXL_20230421_213402619.jpg | Bin .../posts/dna-rna-extractions}/funnel.png | Bin docs/posts/dna-rna-extractions/index.html | 696 +++++++++++ docs/posts/rna-woes/18aug23_drop_plot.jpg | Bin 0 -> 133944 bytes docs/posts/rna-woes/18aug23_drop_table.jpg | Bin 0 -> 225166 bytes docs/posts/rna-woes/index.html | 1045 ++++++++++++++++- docs/posts/rna-woes/nanodrop_plot_03AUG23.jpg | Bin 0 -> 124496 bytes docs/search.json | 46 +- docs/sitemap.xml | 8 +- .../PXL_20230421_213402619.jpg | Bin 0 -> 3018620 bytes .../PXL_20230421_214010536.jpg | Bin .../PXL_20230421_230040973.jpg | Bin .../PXL_20230421_231214054.jpg | Bin .../PXL_20230421_231820714.jpg | Bin posts/dna-rna-extractions/funnel.png | Bin 0 -> 32788 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b/_freeze/posts/rna-woes/index/execute-results/html.json new file mode 100644 index 0000000..be8fef8 --- /dev/null +++ b/_freeze/posts/rna-woes/index/execute-results/html.json @@ -0,0 +1,14 @@ +{ + "hash": "7e64e4d9edec4bbcd55860ab837e080e", + "result": { + "markdown": "---\ntitle: \"The RNA Inhibitors\"\nsubtitle: \"A tragic saga of continued troubles\"\nauthor: \"Sarah Tanja\"\ndate: '08/22/2023'\ncategories: [lab records]\ndraft: false\ntoc: true\ntoc-title: Contents \ntoc-depth: 5\ntoc-location: left\nbibliography: references.bib\nreference-location: margin\ncitation-location: margin\nquarto:\n package_options:\n cran: \"https://cran.rstudio.com/\"\n---\n\n\n\n\n\n\n## So your samples failed library prep ... 😰\n\nAfter shipping 20 samples to the University of Texas at Austin GSAF for TagSeq on June 7th 2023, I got a message back from them on June 28th 2023:\n\n> \"Your samples failed in library prep. All 20 of them, the other samples on the 96 well plate as well as our positive control all worked great. We seen this before and it is almost always an indication that there are some type of inhibitors in your samples and it impacted the prep. Based on the quality of the RNA there is nothing more we can do for them, they will not work in the prep until they have been purified, I am not sure how they were cleaned up after the extraction but it needs to be more rigorous and be certain there are no contaminants in the RNA.\"\n\nAfter initial panic and frustration 😨😭... I started researching reverse transcriptase[^1] inhibitors and reaching out to the [Roberts](https://robertslab.github.io/resources/) and [Putnam](http://putnamlab.com/) Labs to ask for advice (recorded in this Roberts Lab [github issue](https://github.com/RobertsLab/resources/issues/1670)).\n\n[^1]: Reverse transcriptase is an enzyme that transcribes single-stranded RNA into complementary DNA (cDNA) during library prep for RNA sequencing. It was discovered in the 1970's by David Baltimore and Howard Temin when studying retroviruses (checkout this [YouTube video](https://www.youtube.com/watch?app=desktop&v=v9EL8V-6la4) for a brief history on reverse transcriptase, and this [YouTube video](https://www.youtube.com/watch?v=POEDYHKyYDk) for a brief explanation of how it is used to turn mRNA to cDNA).\n\nSo what did I find, and what does this mean for my samples?\n\nThe RNA quantity, measured by Qubit Fluorometer, is good. And the RNA quality, measured by UT GSAF Bioanalyzer, is also good. The issue is that there is *something else* in the sample that is preventing the reverse transcriptase from generating the cDNA.\n\nNanodrop purity targets[^2]:\n\n[^2]: Check out [Assessing RNA quantity and purity with Nanodrop](https://toptipbio.com/the-nanodrop-results-explained/) by TopTipBio\n\n- 260/280 (protein purity) **1.80 - 2.00**\n\n- 260/230 (salts purity) **2.00 - 2.20**\n\n### Zymo OneStep PCR Inhibitor Removal Kit\n\nI first tried purifying sample RNA using the [Zymo OneStep PCR Inhibitor Removal Kit](https://www.zymoresearch.com/collections/onestep-pcr-inhibitor-removal-kits)on a subset of the samples.\n\nI made the tables based off an article by [Little Miss Data](https://www.littlemissdata.com/blog/prettytables)\n\n\n::: {.cell hash='index_cache/html/unnamed-chunk-3_b56c15ea5e8de05e367504d439102ca8'}\n\n```{.r .cell-code}\nlibrary(data.table)\nlibrary(dplyr)\nlibrary(formattable)\nlibrary(tidyverse)\n```\n:::\n\n::: {.cell hash='index_cache/html/unnamed-chunk-4_df6b82ccea88d7258dd8d6281adf86b7'}\n\n```{.r .cell-code}\ncustomGreen0 = \"#DeF7E9\"\ncustomGreen = \"#71CA97\"\ncustomRed = \"#ff7f7f\"\n```\n:::\n\n::: {.cell hash='index_cache/html/unnamed-chunk-5_2c9bb9d8152b6ae3fbdbb201f0b2d867'}\n\n```{.r .cell-code}\nsalts <- formatter(\"span\",\n style = x ~ style(\"font-weight\" = ifelse(x > 2.0, \"bold\", NA),\n \"background-color\" = ifelse(x > 2.0, customGreen, \"inherit\")\n ))\n\nproteins <- formatter(\"span\",\n style = x ~ style(\"font-weight\" = ifelse(x > 1.8, \"bold\", NA),\n \"background-color\" = ifelse(x > 1.8, customGreen, \"inherit\")\n ))\n```\n:::\n\n::: {.cell hash='index_cache/html/unnamed-chunk-6_aa7c8f3499d4c77e4582fcad7694eda3'}\n\n```{.r .cell-code}\nrnaNanodrop <- read_csv(\"RNA-Nanodrop.csv\")\nformattable(rnaNanodrop, \n align = \"l\", # aligns text to the left\n list(\n '260-230'= salts,\n '260-280' = proteins)\n )\n```\n\n::: {.cell-output-display}\n\n \n \n \n \n \n \n \n \n \n \n \n \n\n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n\n
sample-ID date ng-uL A260 A280 260-280 260-230 kit
1-CA2a 8/3/2023 63.88 1.597 0.831 1.92 1.40 OneStepPCRInhibitor
1-CH2a 8/3/2023 26.87 0.672 0.340 1.97 0.79 OneStepPCRInhibitor
1-PA2a 8/3/2023 41.26 1.031 0.498 2.07 1.15 OneStepPCRInhibitor
1-Ea 8/3/2023 44.54 1.114 0.562 1.98 1.25 OneStepPCRInhibitor
1-PH1a 8/3/2023 30.24 0.750 0.362 1.97 1.64 OneStepPCRInhibitor
2-PA1a 8/18/2023 34.73 0.868 0.461 1.88 1.14 RNAClean&Concentrate
2-Ea 8/18/2023 20.28 0.507 0.273 1.86 1.19 RNAClean&Concentrate
2-Ea 8/18/2023 13.78 0.345 0.137 2.52 1.98 RNAClean&Concentrate
3-PA1a 8/18/2023 29.59 0.740 0.372 1.99 1.64 RNAClean&Concentrate
4-CA1a 8/18/2023 34.63 0.866 0.472 1.83 1.16 RNAClean&Concentrate
4-Ea 8/18/2023 16.98 0.424 0.201 2.12 1.77 RNAClean&Concentrate
3-Eb 8/18/2023 22.01 0.550 0.239 2.30 1.47 RNAClean&Concentrate
3-CH2a 8/18/2023 36.81 0.920 0.494 1.86 1.06 RNAClean&Concentrate
1-Ea 8/18/2023 26.94 0.674 0.331 2.04 1.50 RNAClean&Concentrate
3-CA1b 8/18/2023 14.02 0.350 0.148 2.36 1.80 RNAClean&Concentrate
2-CA2a 8/18/2023 39.03 0.976 0.524 1.86 1.07 RNAClean&Concentrate
3-PH1a 8/18/2023 14.13 0.353 0.128 2.76 2.29 RNAClean&Concentrate
2-PH2a 8/18/2023 15.61 0.390 0.181 2.16 1.67 RNAClean&Concentrate
4-PA2a 8/18/2023 28.58 0.715 0.378 1.89 0.98 RNAClean&Concentrate
1-CH2a 8/18/2023 13.93 0.348 0.157 2.22 1.31 RNAClean&Concentrate
1-PH1a 8/18/2023 27.61 0.690 0.320 2.15 1.84 RNAClean&Concentrate
2-CH1b 8/18/2023 18.28 0.457 0.219 2.09 1.46 RNAClean&Concentrate
1-PA2a 8/18/2023 35.33 0.883 0.414 2.13 2.38 RNAClean&Concentrate
4-PH1b 8/18/2023 13.48 0.337 0.139 2.43 1.87 RNAClean&Concentrate
1-CA2a 8/18/2023 42.86 1.072 0.525 2.04 1.93 RNAClean&Concentrate
4-CH1a 8/18/2023 28.95 0.724 0.362 2.00 1.41 RNAClean&Concentrate
\n:::\n:::\n\n\n| sample ID | ng/uL | 260/280 | 260/230 |\n|-----------|-------|---------|---------|\n| 1-CA2a | 63.88 | 1.92 | 1.40 |\n| 1-CH2a | 26.87 | 1.97 | 0.79 |\n| 1-PA2a | 41.26 | 2.07 | 1.15 |\n| 1-Ea | 44.54 | 1.98 | 1.25 |\n| 1-PH1a | 30.24 | 1.97 | 1.64 |\n\n::: {.callout-caution appearance=\"minimal\" icon=\"false\"}\nEven after running samples through the Zymo OneStep PCR Inhibitor Removal Kit, The 260/230 ratios are all below the target of 2.0, indicating contamination by salts.\n:::\n\n![Nanodrop plot results](nanodrop_plot_03AUG23.jpg)\n\n: *RNA Nanodrop absorbance plots after passing samples through Zymo OneStep PCR Inhibitor Removal Kit*\n\n### Zymo RNA Clean & Concentrator Kit\n\nI next tried to purify with the Zymo RNA Clean & Concentrator Kit:\n\n![Nanodrop table results](18aug23_drop_table.jpg)\n\n: *RNA Nanodrop results after passing Zymo DNA/RNA Quick Miniprep Plus extracted samples through the Zymo RNA Clean & Concentrator Kit.*\n\n> \"A 260/280 ratio greater than 2.0 does NOT indicate the presence of especially high quality DNA, it most often reflects residual contaminants, such as phenol .\" -[@zotero-2042]\n>\n> \" ... samples with purity ratios that range from 1.8 to 2.0 are typically considered pure. Although purity ratios are very useful and informative, they do not always provide a complete picture of sample purity.\"\n>\n> \"Residual chemicals from nucleic acid extraction processes (such as guanidine salts or phenol) or cell components that co-extract with the nucleic acids (such as protein) can artificially inflate the A260 value.\"\n\n::: {.callout-note appearance=\"minimal\" icon=\"false\"}\nNote that samples 1Ea, 1CH2a, 1CA2a. 1PA2a & 1PH1a had already been passed through the Zymo OneStep PCR Inhibitor Removal Kit.\n:::\n\n![Nanodrop plot results](18aug23_drop_plot.jpg)\n\n: *RNA Nanodrop absorbance plot after passing Zymo DNA/RNA Quick Miniprep Plus extracted samples through the Zymo RNA Clean & Concentrator Kit.*\n\n### Comparing...\n\n| | | | | | | |\n|-----------|---------|---------|-----------|-----------|---------|--------------|\n| | OneStep | PCR | Inhibitor | RNA Clean | & | Concentrator |\n| sample ID | ng/uL | 260/280 | 260/230 | ng/uL | 260/280 | 260/230 |\n| 1-CA2a | 63.88 | 1.92 | 1.40 | | | |\n| 1-CH2a | 26.87 | 1.97 | 0.79 | | | |\n| 1-PA2a | 41.26 | 2.07 | 1.15 | | | |\n| 1-Ea | 44.54 | 1.98 | 1.25 | | | |\n| 1-PH1a | 30.24 | 1.97 | 1.64 | | | |\n", + "supporting": [], + "filters": [ + "rmarkdown/pagebreak.lua" + ], + "includes": {}, + "engineDependencies": {}, + "preserve": {}, + "postProcess": true + } +} \ No newline at end of file diff --git a/docs/index.html b/docs/index.html index f47dd32..1680eb2 100644 --- a/docs/index.html +++ b/docs/index.html @@ -180,38 +180,34 @@
Categories
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-Daily Lab Log +The RNA Inhibitors

-Enter at your peril; This one is for me, not you +A tragic saga of continued troubles
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-The RNA Inhibitors +Daily Lab Log

-A tragic saga of continued troubles +Enter at your peril; This one is for me, not you