The RNA Inhibitors

A tragic saga of continued troubles

lab records
Author

Sarah Tanja

Published

August 22, 2023

So your samples failed library prep … 😰

After 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:

“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.”

After initial panic and frustration 😨😭… I started researching reverse transcriptase1 inhibitors and reaching out to the Roberts and Putnam Labs to ask for advice (recorded in this Roberts Lab github issue).

  • 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 for a brief history on reverse transcriptase, and this YouTube video for a brief explanation of how it is used to turn mRNA to cDNA).

    • So what did I find, and what does this mean for my samples?

    The 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.

    Assessing Purity

    Nanodrop purity targets2:

  • 2 Check out Assessing RNA quantity and purity with Nanodrop by TopTipBio

    • 260/280 (protein purity) 1.80 - 2.00

    • 260/230 (salts purity) 2.00 - 2.20

    ⚠️ Phenol Contamination may manifest in a 260/280 ratio greater than 2.00 !

    “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.” -(“Ultraviolet Spectrophotometry - an Overview | ScienceDirect Topics” n.d.)

    “Ultraviolet Spectrophotometry - an Overview | ScienceDirect Topics.” n.d. Accessed August 22, 2023. https://www.sciencedirect.com/topics/biochemistry-genetics-and-molecular-biology/ultraviolet-spectrophotometry.

    ” … 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.”

    “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.”

    Zymo OneStep PCR Inhibitor Removal Kit

    I first tried purifying sample RNA using the Zymo OneStep PCR Inhibitor Removal Kit on a subset of the samples.

    I followed the kit instructions, and assessed the samples for purity using a Nanodrop.

    Nanodrop plot results

    RNA Nanodrop absorbance plot after passing Zymo DNA/RNA Quick Miniprep Plus extracted RNA samples through the Zymo OneStep PCR Inhibitor Kit. Run 03AUG2023.

    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-Ea 8/3/2023 44.54 1.114 0.562 1.98 1.25 OneStepPCRInhibitor
    1-PA2a 8/3/2023 41.26 1.031 0.498 2.07 1.15 OneStepPCRInhibitor
    1-PH1a 8/3/2023 30.24 0.750 0.362 1.97 1.64 OneStepPCRInhibitor

    Even 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. Any 260/280 protein ratios OVER 2.0 may indicate contamination by phenols, which absorb between 280-300nm wavelengths. So… be suspicious when you see at 260/280 ratio higher than 2.0!

    The Zymo rep responded with the following advice:

    “The guanidine salts in our DNA/RNA Shield and DNA/RNA Lysis Buffer could be the reason of increased A230. We see this happening most often when small impurities (e.g., cell debris, protein aggregation) were transferred from sample processing and got stuck on the column. They would absorb buffers and release them during elution. Ways to avoid this include increasing the amount of DNA/RNA Shield used during processing, reducing sample input, and transferring only a fraction of sample (e.g.,500 µl from 800 µl) from the BashingBead tube to avoid debris. You can also further purify your existing eluted samples with either the RNA Clean & Concentrator that is on the way to you, or by following the”Liquids/Reaction Clean-up” procedure included in the Appendices section Quick-DNA/RNA Miniprep Plus’s protocol.”

    Zymo RNA Clean & Concentrator Kit

    I next tried to purify the RNA with the Zymo RNA Clean & Concentrator Kit:

    Nanodrop plot results

    RNA Nanodrop absorbance plot after passing Zymo DNA/RNA Quick Miniprep Plus extracted RNA samples through the Zymo RNA Clean & Concentrator Kit. Run 18AUG2023.

    sample-ID date ng-uL A260 A280 260-280 260-230 kit
    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

    P.S. I made these pretty tables based off this article by Little Miss Data

    The 260/280 ratios over 2.0 may indicate worse phenol contamination!

    Did the RNA Clean & Concentrate Kit improve the samples that had already been through the OneStep PCR Inhibitor Removal Kit?

    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-Ea 8/3/2023 44.54 1.114 0.562 1.98 1.25 OneStepPCRInhibitor
    1-PA2a 8/3/2023 41.26 1.031 0.498 2.07 1.15 OneStepPCRInhibitor
    1-PH1a 8/3/2023 30.24 0.750 0.362 1.97 1.64 OneStepPCRInhibitor

    Nanodrop Results from OneStep PCR Inhibitor Removal

    sample-ID date ng-uL A260 A280 260-280 260-230 kit
    1-CA2a 8/18/2023 42.86 1.072 0.525 2.04 1.93 RNAClean&Concentrate
    1-CH2a 8/18/2023 13.93 0.348 0.157 2.22 1.31 RNAClean&Concentrate
    1-Ea 8/18/2023 26.94 0.674 0.331 2.04 1.50 RNAClean&Concentrate
    1-PA2a 8/18/2023 35.33 0.883 0.414 2.13 2.38 RNAClean&Concentrate
    1-PH1a 8/18/2023 27.61 0.690 0.320 2.15 1.84 RNAClean&Concentrate

    Nanodrop Results from RNA Clean & Concentrate

    The RNA Clean & Concentrator improved the 260/230 ratios for samples 1Ea, 1CH2a, 1CA2a. 1PA2a & 1PH1a which had already been passed through the Zymo OneStep PCR Inhibitor Removal Kit.

    The 260/280 ratios are suspiciously high, making me wonder about phenol contamination... However, the Zymo Kit is phenol-free, so the only source of phenol would be from the samples themselves. We’ll find out for sure when we send in the purified samples for another attempt at TagSeq Library Prep by UT GSAF.

    RNA Clean & Concentrator follow-up tests

    24-AUG-2023

    Samples:

    • 10-Aa
    • 6-Aa
    sample-ID date ng-uL A260 A280 260-280 260-230
    10AaDNA 8/24/2023 177.55 3.55 1.89 1.87 1.02
    6AaDNA 8/24/2023 140.97 2.81 1.49 1.89 1.84
    10AaRNA 8/24/2023 41.71 1.04 0.49 2.11 2.19
    6AaRNA 8/24/2023 56.00 1.40 0.63 2.20 2.05

    31-AUG-2023

    In this extraction, I accidentally eluted the RNA into the collection tube instead of a nuclease free tube in the RNA Clean & Concentrator elution step. This might have contaminated the eluted RNA, so I took all 10 samples and ran them through another round of RNA Clean & Concentrator prior to quantification on Nanodrop & Qubit.

    Samples:

    • mock
    • blank
    • 4-LA2a
    • 1-HH2a
    • 2-HH2a
    • 3-HA2a
    • 3-MA2a
    • 1-HA2a
    • 4-MA2a
    • 3-HH2a
    # Sample ID User name Date and Time Nucleic Acid Unit A260 (Abs) A280 (Abs) 260/280 260/230 Sample Type Factor
    1 3HA2a medusaFTIR 8/31/2023 1:46:40 PM 454.2 ng/µl 9.084 4.905 1.85 2.08 DNA 50
    2 NA medusaFTIR 8/31/2023 1:07:32 PM -0.2 ng/µl -0.004 -0.008 0.56 0.28 DNA 50
    3 4LA2a medusaFTIR 8/31/2023 1:42:30 PM 467.8 ng/µl 9.356 5.051 1.85 2.23 DNA 50
    4 3MA2a medusaFTIR 8/31/2023 1:44:46 PM 268.0 ng/µl 5.361 2.891 1.85 2.16 DNA 50
    5 blank medusaFTIR 8/31/2023 1:50:03 PM 11.4 ng/µl 0.229 0.149 1.54 0.87 DNA 50
    6 mock medusaFTIR 8/31/2023 1:48:14 PM 7.0 ng/µl 0.140 0.117 1.20 0.42 DNA 50
    7 1HA2a medusaFTIR 8/31/2023 1:51:45 PM 332.7 ng/µl 6.653 3.579 1.86 2.26 DNA 50
    8 4MA2a medusaFTIR 8/31/2023 1:58:22 PM 126.7 ng/µl 2.534 1.397 1.81 1.92 DNA 50
    9 2HH2a medusaFTIR 8/31/2023 1:59:59 PM 9.9 ng/µl 0.198 0.151 1.32 0.71 DNA 50
    10 2HH2a medusaFTIR 8/31/2023 2:01:15 PM 10.4 ng/µl 0.208 0.152 1.36 0.70 DNA 50
    11 1HH2a medusaFTIR 8/31/2023 2:02:35 PM 224.5 ng/µl 4.490 2.443 1.84 1.95 DNA 50
    12 3HH2a medusaFTIR 8/31/2023 2:05:22 PM 213.0 ng/µl 4.261 2.325 1.83 2.02 DNA 50
    13 4MA2a medusaFTIR 8/31/2023 2:16:38 PM 14.7 ng/µl 0.368 0.209 1.76 2.46 RNA 40
    14 4LA2a medusaFTIR 8/31/2023 2:18:13 PM 70.4 ng/µl 1.760 0.945 1.86 1.16 RNA 40
    15 3HA2a medusaFTIR 8/31/2023 2:19:55 PM 78.4 ng/µl 1.959 1.183 1.66 0.89 RNA 40
    16 2HH2a medusaFTIR 8/31/2023 2:21:39 PM 12.3 ng/µl 0.307 0.156 1.97 2.46 RNA 40
    17 1HH2a medusaFTIR 8/31/2023 2:23:07 PM 66.4 ng/µl 1.661 0.831 2.00 2.39 RNA 40
    18 1HA2a medusaFTIR 8/31/2023 2:24:28 PM 45.2 ng/µl 1.131 0.568 1.99 2.20 RNA 40
    19 3MA2a medusaFTIR 8/31/2023 2:26:15 PM 35.5 ng/µl 0.887 0.480 1.85 1.63 RNA 40
    20 3HH2a medusaFTIR 8/31/2023 2:27:42 PM 29.8 ng/µl 0.745 0.377 1.97 2.36 RNA 40

    Plots of RNA nucleic acid absorbance spectra for 10 samples

    RNA Nanodrop absorbance plots after conducting modified extraction protocol and passing samples through two rounds of RNA Clean & Concentrator

    The absorbance spectra show that samples 4-LA2a, 3-MA2a & 3-HA2a have salts contamination and 3-HA2a shows protein contamination. In the table they have 260/280 and 260/230 values below the desired targets. The other 5 RNA samples seem to pass the contamination check… as far as we can tell….

    06-SEP-2023

    These 12 samples were passed through the Zymo RNA Clean & Concentrator kit a 2nd (or 3rd!) time to see if doing the kit another round made further improvements to purity

    Samples:

    • 1-CA2a
    • 1-CH2a
    • 1-Ea
    • 1-PH1a
    • 2-CH1b
    • 3-CH2a
    • 3-MA2a
    • 3-HA2a
    • 4-CH1a
    • 4-MA2a
    • 4-PH1b
    • 4-LA2a

    I wasn’t stoked on the results….

    Nanodrop of RNA from 06SEP2023

    Sample ID qbit1 qbit2 qbitAVG # Date and Time Nucleic Acid Unit A260 (Abs) A280 (Abs) 260/280 260/230 Sample Type
    3HA2a 25.8 25.0 25.4 8 9/6/2023 3:51:11 PM 50.4 ng/µl 1.261 0.618 2.04 2.08 RNA
    2CH1b 0.0 0.0 0.0 15 9/6/2023 4:02:34 PM 26.8 ng/µl 0.671 0.376 1.78 1.04 RNA
    3MA2a 30.0 29.6 29.8 11 9/6/2023 3:56:02 PM 99.9 ng/µl 2.497 1.449 1.72 0.94 RNA
    1CA2a 22.6 21.8 22.2 7 9/6/2023 3:49:41 PM 49.3 ng/µl 1.234 0.635 1.94 2.15 RNA
    1Ea 12.0 11.0 11.5 9 9/6/2023 3:52:29 PM 66.3 ng/µl 1.658 1.006 1.65 0.80 RNA
    3CH2a 16.8 16.6 16.7 12 9/6/2023 3:57:46 PM 21.5 ng/µl 0.538 0.261 2.06 2.42 RNA
    4CH1a 10.8 10.4 10.6 14 9/6/2023 4:01:15 PM 50.2 ng/µl 1.254 0.720 1.74 0.98 RNA
    1CH2a 11.6 11.0 11.3 16 9/6/2023 4:04:05 PM 9.5 ng/µl 0.239 0.118 2.03 1.45 RNA
    1PH1a 13.8 12.6 13.2 13 9/6/2023 3:59:37 PM 27.4 ng/µl 0.684 0.339 2.02 2.28 RNA
    4LA2a 31.0 30.2 30.6 10 9/6/2023 3:54:21 PM 82.6 ng/µl 2.065 1.052 1.96 1.42 RNA
    4MA2a 13.0 12.4 12.7 4 9/6/2023 3:45:27 PM 22.1 ng/µl 0.553 0.266 2.08 2.01 RNA
    4PH1b 10.0 13.2 11.6 5 9/6/2023 3:47:09 PM 40.2 ng/µl 1.004 0.604 1.66 0.80 RNA
    4PH1b 10.0 13.2 11.6 6 9/6/2023 3:48:15 PM 20.4 ng/µl 0.510 0.279 1.83 1.18 RNA
    blank NA NA NA 1 9/6/2023 3:40:55 PM -19.2 ng/µl -0.481 -0.275 1.75 1.90 RNA
    blank(Reblank) NA NA NA 2 9/6/2023 3:42:29 PM -0.1 ng/µl -0.001 -0.009 0.16 0.07 RNA
    blank NA NA NA 3 9/6/2023 3:43:25 PM -0.6 ng/µl -0.015 -0.021 0.72 27.77 RNA

    I want to pick out the samples that are the best purity to send to UT Austin GSAF to see if they will pass library prep.

    The purified RNA samples that are ‘green across the board’ on both concentration and purity and are ‘good to go’ are:

    • 3-HA2a (50.4ng/uL nanodrop | 25.4 qubit)

    • 1-CA2a (49.3ng/uL nanodrop | 22.2 qubit)

    The ones that are borderline…..

    • 1-PH1a (27.4ng/uL nanodrop | 13.2 qubit?)

    • 3-CH2a (21.5ng/uL nanodrop | 16.7 qubit?)

    • 4-MA2a (22.1ng/uL nanodrop | 12.7 qubit?)

    • 4-LA2a ( 260/230: 1.42? | 30.6 qubit)

    • 10-Aa?

    • 6Aa?

    Some of the sample purity was made WORSE by running them through RNA Clean & Concentrator a second or third time….

    3MA2a on 31AUG2023:

    260/280: 1.85

    260/230: 1.63

    vs.

    3MA2a on 06SEP2023:

    260/280: 1.72

    260/230: 0.94

    However other samples were made slightly better…

    3HA2a on 31AUG2023

    260/280: 1.85

    260/230: 2.08

    vs.

    3HA2a on 06SEP2023

    260/280: 2.04

    260/230: 2.08

    Really still frustrated that I’m not getting consistent results… Does this kit work to purify the samples? Not in large batches…. few of the samples meet the purity targets. I’m also worried that the more I work with the RNA the more it degrades and I’m losing quantity (ng/uL) and possibly the quality (time will tell when the RIN scores come back from UT GSAF)