NIH, National Cancer Institute, Division of Cancer Treatment and Diagnosis (DCTD) NIH - National Institutes of Health National Cancer Institute DCTD - Division of Cancer Treatment and Diagnosis

Stool metatranscriptomics: A technical guideline for mRNA stabilisation and isolation.

Author(s): Reck M, Tomasch J, Deng Z, Jarek M, Husemann P, Wagner-Döbler I

Publication: BMC Genomics, 2015, Vol. 16, Page 494

PubMed ID: 26140923 PubMed Review Paper? No

Purpose of Paper

This paper compared the yield and purity of RNA isolated from stool by four different isolation methods and compared the integrity of RNA among matched specimens that were stored for up to 15 days in RNAlater, All Protect, DNA stabilizer, and RNA Protect.  Sequencing metrics and results were compared among specimens stored for up to 6 days in RNAlater (at room temperature and 4°C), in RNA Protect or frozen.

Conclusion of Paper

The highest RNA yields were obtained from stool specimens when the phenol-chloroform-based method was used, followed by the PowerMicrobiome Kit, and the Stool Total RNA Purification Kit (modified followed by original), and lowest using the RNeasy mini Kit. Each RNA isolation method evaluated had an acceptable absorbance ratio at 260 to 280 nm, although the PowerMicrobiome kit generated a higher 260/230 ratio than the other kits evaluated. The PowerMicrobiome Kit was selected by the authors for subsequent analysis given its superior purity, speed, and reproducibility.  

Storage of fecal specimens resulted in a decline in RNA integrity number (RIN), although the decline was slowest in specimens preserved in RNAlater-followed by All Protect and DNA stabilizer; RIN was least stable when RNA Protect was used as a preservative. Comparatively, RNA from stool specimens preserved with and stored in RNAlater had fewer detectable copy numbers of endogenous and spike-in RNA, fewer read counts, fewer taxonomically assignable reads, and a smaller percentage of rRNA than those preserved with and stored in RNA Protect., some differences in the taxonomic profile were observed between RNAlater and frozen specimens in the 0 h storage control, and these differences were larger than those between frozen specimens and specimens stored in RNA Protect for <24 h.

Studies

  1. Study Purpose

    This study compared the yield and purity of RNA isolated from stool using four different isolation methods. A single fecal specimen was sampled at three locations, mixed with stabilizer solution for 5 min at room temperature, and then frozen at -80°C. The specimens were thawed on ice, homogenized with ice cold PBS on a vortex, and centrifuged for 1 min at 700 g at 4°C, and cells were then pelleted from the specimens by centrifugation at 9000 g at 4°C. The cells were resuspended in lysis buffer and RNA was extracted with the Stool Total RNA Purification Kit using the supplied glass beads or a modification using 0.1 mm zirconia beads, the Power microbiome RNA Isolation Kit, the RNeasy Mini Kit, and a modified phenol-chloroform procedure. RNA was quantified by spectrophotometer.

    Summary of Findings:

    The highest RNA yields were obtained from stool specimens extracted with the phenol-chloroform-based method (mean yield ~ 63 µg), followed by the PowerMicrobiome Kit (mean yield ~ 34 µg), the Stool Total RNA Purification Kit (mean yield ~ 25 µg), and the RNeasy mini Kit (mean yield ~7 µg).  All RNA isolated (regardless of extraction method)  had an absorbance ratio at 260 to 280 that approach 2.0, indicating effective removal of proteins. The 260/230 ratio was higher when the PowerMicrobiome Kit was used for extraction than the other kits evaluated, indicating a lower level of contamination due to salts and solvents.  The authors opted to use the PowerMicrobiome Kit for subsequent studies given its superior purity, speed, and reproducibility.

    Biospecimens
    Preservative Types
    • RNAlater
    • Frozen
    Diagnoses:
    • Normal
    Platform:
    AnalyteTechnology Platform
    RNA Spectrophotometry
    Pre-analytical Factors:
    ClassificationPre-analytical FactorValue(s)
    Analyte Extraction and Purification Analyte isolation method Stool Total RNA Purification Kit using the supplied glass beads
    Stool Total RNA Purification Kit using 0.1 mm zirconia beads
    Power microbiome RNA Isolation Kit
    RNeasy Mini Kit
    Modified phenol-chloroform procedure
  2. Study Purpose

    This study compared the integrity of RNA isolated from matched stool specimens that were stored for up to 15 days in RNAlater, All Protect, DNA stabilizer, and RNA Protect; sequencing metrics and results were compared among stool specimens stored in RNAlater at room temperature and 4°C, and among stool specimens stored in RNA Protect or frozen (-80°C) for up to 6 days. A single fecal specimen was sampled at three locations, and the three subsamples were mixed before preservation with RNA Protect, RNALater, Allprotect, or DNA stabilizer. E. coli expressing Cherry and GFP was added to all specimens. The specimens were stored for 0, 2, 6, 12, 24, 144 (6 days), and 360 h at 4°C and/or room temperature (RNAlater only).  At each timepoint, an aliquot was removed and frozen at -80°C.  The specimens were thawed on ice, homogenized with ice-cold PBS on a vortex, and centrifuged for 1 min at 700 g at 4°C, and cells were then pelleted from the specimens by centrifugation at 9000 g at 4°C. The cells were resuspended in lysis buffer before extraction with the PowerMicrobiome RNA Isolation Kit and Ribozero treatment. RNA integrity was assessed by Bioanalyzer. Levels of mCherry, GFP, 23s rRNA, and GAPDH were quantified by real-time PCR.  Sequencing libraries were constructed using RNA isolated from specimens preserved with RNAlater and RNA Protect Script Seq Illumina Kit and sequenced using a HiSeq 2000 Sequencer. Taxonomic classification was performed using Kraken and bwa v. 0.7.5 with SAMtools.

    Summary of Findings:

    At the 0 h storage control timepoint, RNA isolated from all stool specimens had a RIN > 9, but only specimens preserved with RNAlater maintained a RIN >7 after 15 days (360 h) at room temperature and a RIN >8.5 after 15 days at 4°C. While not as effective as RNAlater, Allprotect also stabilized RNA, as RINs close to 7 were observed after 6 days at room temperature. In contrast, RNA degraded rapidly when stool specimens were preserved with RNA Protect at room temperature (RIN =3 after 6 days), or DNA stabilizer at room temperature (RIN =5 after 6 days). While real-time PCR quantification of both spike-in controls was comparable among stool specimens preserved in RNA Protect at room temperature or RNAlater at either temperature for ≤24 h or when short primers were used, copy number declined faster in stool specimens preserved with RNA Protect that were stored for >24 h. Read counts from the RNAlater-preserved stool specimens were unaffected by storage duration or storage temperature, but the specimens stored for 24 h and 144 h in RNA Protect had higher read counts than the 0 h control specimen (82 and 93 million reads, respectively, versus 72 million reads). Importantly, the RNA Protect specimens stored for 24 and 144 h also had a much higher percentage of rRNA compared to other specimens (40% and 69%, respectively versus 2-16%), which the authors state is likely the result of RiboZero reliance on intact 5’ and 3’ ends. Further, the number of taxonomically assignable reads was stable in RNAlater-preserved stool specimens stored at 4°C for 6 d at room temperature but declined with storage in RNA Protect-preserved specimens or the frozen specimen. Interestingly, a decline in assignable reads was also observed for the frozen specimen.  The taxonomic composition of the specimens was very strongly correlated between timepoints for all specimens, but correlations were stronger for specimens preserved in RNAlater than RNA Protect. Importantly, some differences in the taxonomic profile were observed between RNAlater and frozen specimens at the 0 h storage control time point, and these differences were larger than those between the frozen specimen and specimens stored in RNA Protect for <24 h. In specimens preserved by RNA Protect, the transcripts that were most affected by storage fell under the category “transcription”.  Stool specimens preserved by RNA Protect and RNAlater had similar recovery rates for mCherry (14.7% and 17.8%, respectively) and sFGFP (8.7% and 9.7%, respectively), and the RNA was mostly localized with gram-negative bacteria. Sequencing confirmed that declines of mCherry and sFGFP were small in specimens stored in RNAlater but loss of >50% of copies in specimens stored in RNA Protect. While Kraken and bwa both identified Firmicutes and Bacteriodetes as the most common phyla, there were differences in the relative abundances of the families identified.

    Biospecimens
    Preservative Types
    • RNAlater
    • Frozen
    Diagnoses:
    • Normal
    Platform:
    AnalyteTechnology Platform
    RNA Automated electrophoresis/Bioanalyzer
    RNA Next generation sequencing
    RNA Real-time qRT-PCR
    Pre-analytical Factors:
    ClassificationPre-analytical FactorValue(s)
    Biospecimen Preservation RNA stabilization method RNAlater
    RNA Protect
    All protect
    DNA stabilizer
    Storage Storage duration 0 h
    2 h
    6 h
    12 h
    24 h
    144 h (6 days)
    360 h (15 days)
    Storage Storage temperature Room temperature (RNAlater)
    4°C
    Next generation sequencing Specific Data handling bwa
    Kraken

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