CoChat
Sign Up
Cited by 53Molecular Biology Techniques and ApplicationsMicroRNA in disease regulationGene expression and cancer classification

MicroRNA expression in formalin‐fixed paraffin embedded tissue using real time quantitative PCR: the strengths and pitfalls

Open access
Dijkstra, J. R., Mekenkamp, L. J. M., Teerenstra, S., De Krijger, I., Nagtegaal, I. D.·2012·Journal of Cellular and Molecular Medicine
DOI 10.1111/j.1582-4934.2011.01467.xPMC PMC3822839
Open this paper in CoChat — free accountOpen PDF

Abstract

Introduction RNA isolation from formalin‐fixed paraffin‐embedded (FFPE) tissue RNA quality control cDNA synthesis Real Time Quantitative PCR Reference genes Data analysis Conclusions MicroRNAs (miRNAs) are a group of small non‐coding RNAs with a huge impact in a wide range of biological processes, including cancer. The evidence collected to date demonstrates that miRNAs represent valid diagnostic, prognostic and predictive markers in cancer. The identification of these miRNA biomarkers in archived tissues has been facilitated by novel development and refinement of detection methodologies. Quantitative real‐time reverse‐transcription PCR (qRT‐PCR) is one of the most common methods used to detect low levels of miRNAs with high sensitivity and specificity. However, several technical parameters should be identified and optimized in order to obtain meaningful and reproducible results. The purpose of this review is to describe some of these technical parameters and improve the validity and reliability of miRNA expression studies.

The claims and statistics of this paper haven’t been checked yet.

Run the check — free, no account needed. It reads the open-access text and checks the sources it cites.

A few free checks a day · papers already verified open instantly · results are public

Integrity signals

Registry

Crossref · 10.1111/j.1582-4934.2011.01467.x

Cites

5 of 5 references verified0 citation errors · 0 not checkable

Statistics

Claims the paper makes

The claims and statistics of this paper haven’t been checked yet.

Run the check — free, no account needed. It reads the open-access text and checks the sources it cites.

A few free checks a day · papers already verified open instantly · results are public

Claims other papers make about this one

Each is checked against this paper’s own text: does it say what the citing paper says it does?

  • All reactions for the study were pipetted into a single 96-well plate each for ddPCR and qPCR to eliminate inter-plate variability.

    “Therefore, all reactions for the study were pipetted into a single 96-well plate each for ddPCR and qPCR technologies.”

    From Droplet Digital PCR versus qPCR for gene expression analysis with low abundant targets: from variable nonsense to publication quality data (2017) · cites this paper as [17]

    The Quantitative real-time reverse transcription PCR section says that inter-plate variability can increase variation and that keeping the relevant qRT-PCR assays or samples on one plate removes the need for inter-plate correction. It does not say that this study pipetted all reactions into single 96-well plates, or extend the advice to ddPCR. No page number is available in our copy. (LLM, conf=0.85)

    Experimental — accuracy not yet measured

Self-check

Percentages, sums and reported differences re-computed from the paper’s own numbers.

The self-check needs the paper’s full text.

Cites

Flagged first, then by citations

Research works printed but not linked: 39

  1. [1]Bartel DP MicroRNAs: genomics, biogenesis, mechanism, and function Cell 2004 116 281 97 14744438
  2. [2]Lewis BP Burge CB Bartel DP Conserved seed pairing, often flanked by adenosines, indicates that thousands of human genes are microRNA targets Cell 2005 120 15 20 15652477
  3. [3]Farazi TA Spitzer JI Morozov P miRNAs in human cancer J Pathol 2011 223 102 15 21125669 PMC3069496
  4. [4]Volinia S Calin GA Liu CG A microRNA expression signature of human solid tumours defines cancer gene targets Proc Natl Acad Sci U S A 2006 103 2257 61 16461460 PMC1413718
  5. [5]Yan LX Huang XF Shao Q MicroRNA miR-21 overexpression in human breast cancer is associated with advanced clinical stage, lymph node metastasis and patient poor prognosis RNA 2008 14 2348 60 18812439 PMC2578865
  6. [6]Schetter AJ Leung SY Sohn JJ MicroRNA expression profiles associated with prognosis and therapeutic outcome in colon adenocarcinoma JAMA 2008 299 425 36 18230780 PMC2614237
  7. [7]Chan JA Krichevsky AM Kosik KS MicroRNA-21 is an antiapoptotic factor in human glioblastoma cells Cancer Res 2005 65 6029 33 16024602
  8. [9]Koshiol J Wang E Zhao Y Strengths and limitations of laboratory procedures for microRNA detection Cancer Epidemiol Biomarkers Prev 2010 19 907 11 20332265 PMC2852469
  9. [10]Derveaux S Vandesompele J Hellemans J How to do successful gene expression analysis using real-time PCR Methods 2010 50 227 30 19969088
  10. [11]Benes V Castoldi M Expression profiling of microRNA using real-time quantitative PCR, how to use it and what is available Methods 2010 50 244 9 20109550
  11. [12]Lewis F Maughan NJ Smith V Unlocking the archive-gene expression in paraffin-embedded tissue J Pathol 2001 195 66 71 11568892
  12. [13]Masuda N Ohnishi T Kawamoto S Analysis of chemical modification of RNA from formalin-fixed samples and optimization of molecular biology applications for such samples Nucleic Acids Res 1999 27 4436 43 10536153 PMC148727
  13. [14]Klopfleisch R Weiss AT Gruber AD Excavation of a buried treasure-DNA, mRNA, miRNA and protein analysis in formalin fixed, paraffin embedded tissues Histol Histopathol 2011 26 797 810 21472693
  14. [15]Godfrey TE Kim SH Chavira M Quantitative mRNA expression analysis from formalin-fixed, paraffin-embedded tissues using 5′ nuclease quantitative reverse transcription-polymerase chain reaction J Mol Diagn 2000 2 84 91 11272893 PMC1906896
  15. [16]Xi Y Nakajima G Gavin E Systematic analysis of microRNA expression of RNA extracted from fresh frozen and formalin-fixed paraffin-embedded samples RNA 2007 13 1668 74 17698639 PMC1986820
  16. [17]Szafranska AE Davison TS Shingara J Accurate molecular characterization of formalin-fixed, paraffin-embedded tissues by microRNA expression profiling J Mol Diagn 2008 10 415 23 18687792 PMC2518736
  17. [18]Doleshal M Magotra AA Choudhury B Evaluation and validation of total RNA extraction methods for microRNA expression analyses in formalin-fixed, paraffin-embedded tissues J Mol Diagn 2008 10 203 11 18403610 PMC2329784
  18. [19]Li J Smyth P Flavin R Comparison of miRNA expression patterns using total RNA extracted from matched samples of formalin-fixed paraffin-embedded (FFPE) cells and snap frozen cells BMC Biotechnol 2007 7 36 17603869 PMC1914054
  19. [20]Glud M Klausen M Gniadecki R MicroRNA expression in melanocytic nevi: the usefulness of formalin-fixed, paraffin-embedded material for miRNA microarray profiling J Invest Dermatol 2009 129 1219 24 19005486
  20. [21]Liu A Tetzlaff MT Vanbelle P MicroRNA expression profiling outperforms mRNA expression profiling in formalin-fixed paraffin-embedded tissues Int J Clin Exp Pathol 2009 2 519 27 19636399 PMC2713447
  21. [22]Nelson PT Wang WX Wilfred BR Technical variables in high-throughput miRNA expression profiling: much work remains to be done Biochim Biophys Acta 2008 1779 758 65 18439437 PMC2660892
  22. [23]Sambrook J Fritsch EF Maniatis T Molecular cloning: a laboratory manual 1989 2 New York Cold Spring Harbor Laboratory Press
  23. [25]Fleige S Walf V Huch S Comparison of relative mRNA quantification models and the impact of RNA integrity in quantitative real-time RT-PCR Biotechnol Lett 2006 28 1601 13 16900335
  24. [26]Jung M Schaefer A Steiner I Robust microRNA stability in degraded RNA preparations from human tissue and cell samples Clin Chem 2010 56 998 1006 20378769
  25. [27]Yu SL Chen HY Yang PC Unique MicroRNA signature and clinical outcome of cancers DNA Cell Biol 2007 26 283 92 17504024
  26. [28]Becker C Hammerle-Fickinger A Riedmaier I mRNA and microRNA quality control for RT-qPCR analysis Methods 2010 50 237 43 20079844
  27. [31]Chen C Ridzon DA Broomer AJ Real-time quantification of microRNAs by stem-loop RT-PCR Nucleic Acids Res 2005 33 e179 16314309 PMC1292995
  28. [32]Lao K Xu NL Yeung V Multiplexing RT-PCR for the detection of multiple miRNA species in small samples Biochem Biophys Res Commun 2006 343 85 9 16529715
  29. [33]Mestdagh P Feys T Bernard N High-throughput stem-loop RT-qPCR miRNA expression profiling using minute amounts of input RNA Nucleic Acids Res 2008 36 e143 18940866 PMC2588502
  30. [34]Tichopad A Kitchen R Riedmaier I Design and optimization of reverse-transcription quantitative PCR experiments Clin Chem 2009 55 1816 23 19643838
  31. [35]Zipper H Brunner H Bernhagen J Investigations on DNA intercalation and surface binding by SYBR Green I, its structure determination and methodological implications Nucleic Acids Res 2004 32 e103 15249599 PMC484200
  32. [36]Whitman DF Dunbar SA Real-time polymerase chain reaction detection methods Recent Pat DNA Gene Seq 2008 2 20 6 19075941
  33. [37]Peltier HJ Latham GJ Normalization of microRNA expression levels in quantitative RT-PCR assays: identification of suitable reference RNA targets in normal and cancerous human solid tissues RNA 2008 14 844 52 18375788 PMC2327352
  34. [38]Chang KH Mestdagh P Vandesompele J MicroRNA expression profiling to identify and validate reference genes for relative quantification in colorectal cancer BMC Cancer 2010 10 173 20429937 PMC2873395
  35. [39]Vandesompele J De Preter K Pattyn F Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes Genome Biol 2002 3: RESEARCH0034 PMC126239 12184808
  36. [40]Meyer SU Pfaffl MW Ulbrich SE Normalization strategies for microRNA profiling experiments: a ‘normal’ way to a hidden layer of complexity? Biotechnol Lett 2010 32 1777 88 20703800
  37. [41]Mestdagh P Van Vlierberghe P De Weer A A novel and universal method for microRNA RT-qPCR data normalization Genome Biol 2009 10 R64 19531210 PMC2718498
  38. [42]Bissels U Wild S Tomiuk S Absolute quantification of microRNAs by using a universal reference RNA 2009 15 2375 84 19861428 PMC2779673
  39. [43]Schmittgen TD Livak KJ Analyzing real-time PCR data by the comparative C(T) method Nat Protoc 2008 3 1101 8 18546601

Cited by

Sorted by citations

Authors
Dijkstra, J. R., Mekenkamp, L. J. M., Teerenstra, S., De Krijger, I., & Nagtegaal, I. D.
Affiliations
Radboud University Medical Center (NL)
Corresponding author
Dijkstra, J. R.
ORCID
Venue
Journal of Cellular and Molecular Medicine
Publisher
Wiley
Published
2012-04
Volume
16
Issue
4
Pages
683-690
ISSN
  • 1582-1838 (ISSN-L)
  • 1582-4934
DOI
10.1111/j.1582-4934.2011.01467.x
PMC
PMC3822839
OpenAlex
W2006635242
Cited by
53
References
5
Citations by year
2026: 3 · 2024: 1 · 2023: 2 · 2021: 2 · 2020: 3 · 2019: 4 · 2018: 8 · 2017: 5 · 2016: 3 · 2015: 6 · 2014: 8 · 2013: 7 · 2012: 1
FWCI
0.46
Type
journal article
Language
en
Open access
Licence
onlinelibrary.wiley.com/termsAndConditions#vor (vor, 15 d)
Locations
  • Journal of Cellular and Molecular Medicine (publishedVersion)
  • Europe PMC (PubMed Central) (publishedVersion)
  • PubMed Central (publishedVersion)
  • PubMed (publishedVersion)
  • Radboud Repository (Radboud University) (submittedVersion)
  • Radboud Repository (Radboud University) (submittedVersion, other-oa)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Molecular Biology Techniques and Applications
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Cancer Research › MicroRNA in disease regulation
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Gene expression and cancer classification
Keywords
microRNA expression, formalin-fixed paraffin-embedded tissue, RNA extraction, RNA quality control, cDNA synthesis, reference genes, microRNA biomarkers, tumor markers, archival tissue, assay reproducibility
MeSH terms
Formaldehyde, Quality Control, RNA, Paraffin Embedding, DNA, Complementary, Reverse Transcriptase Polymerase Chain Reaction, MicroRNAs, Real-Time Polymerase Chain Reaction

About these checks

Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.

Statistics and arithmetic are recomputed, not judged.

Confidence bands are shown only for fields calibrated on our annotated test set. None is calibrated yet, so no band is shown.

What counts as a claim ›How we measure confidence ›How we check this ›

Are you an author, or do you see a problem with this page? Request a review