Application Note

Archiving histological tissue samples by formalin-fixing and embedding in paraffin blocks has been a routine practice for medical and research institutions for many years and large collections of these samples now exist throughout the world.  Estimates of sample numbers range from 300-400 million in the US alone covering virtually all known diseases. This number may be growing by tens of millions of samples per year.1

What makes these samples of particular interest is the rich patient data and medical history associated with each sample.  Because the samples are so well annotated, they are proving to be a valuable source of information for retrospective clinical studies of human diseases.

Initially, it was unclear if the nucleic acids would remain in sufficient condition to provide robust analytical results due to the adverse effects of formalin fixation.  But studies have shown that although degradation to large RNA can occur, in general, small RNAs are unaffected by the fixation process2.  Indeed there are now a multitude of commercially available kits and methods for extracting microRNA from FFPE prepared tissue samples.3

Furthermore, it has been demonstrated in multiple studies that miRNA is minimally affected by FFPE treatment and microRNA extracted from FFPE samples provides reliable expression levels by direct comparison with frozen tissue samples.4,5,6,7,8 MicroRNA expression data was found to be reliable for FFPE samples stored for up to 30 years.9

Example Data:  FFPE vs Frozen Sample Expression Results

Kits for extraction of microRNA from FFPE prepared tissue samples:

  • RecoverAll Total Nucleic Acid Isolation Kit for FFPE – Ambion
  • miRNeasy FFPE Kit (50) – Qiagen
  • FFPE RNA Purification Kit – Norgen

LC Sciences Customer Publications

  1. Magrelli A, Azzalin G, Salvatore M, Viganotti M, Tosto F, Colombo T, Devito R, Di Masi A, Antoccia A, Lorenzetti S, Maranghi F, Mantovani A, Tanzarella C, Macino G, Taruscio D. (2009) Altered microRNA Expression Patterns in Hepatoblastoma Patients. Transl Oncol 2(3), 157-63. [article]
  2. Zhao Y, Deng C, Wang J, Xiao J, Gatalica Z, Recker RR, Xiao GG. (2010) Let-7 family miRNAs regulate estrogen receptor alpha signaling in estrogen receptor positive breast cancer. Breast Cancer Res Treat 127(1), 69-80. [article]
  3. Zhu Y, Yu X, Fu H, Wang H, Wang P, Zheng X, Wang Y. (2010) MicroRNA-21 is involved in ionizing radiation-promoted liver carcinogenesis. Int J Clin Exp Med 3(3), 211-22. [article]
  4. White NM, Khella HW, Grigull J, Adzovic S, Youssef YM, Honey RJ, Stewart R, Pace KT, Bjarnason GA, Jewett MA, Evans AJ, Gabril M, Yousef GM. (2011) miRNA profiling in metastatic renal cell carcinoma reveals a tumour-suppressor effect for miR-215. Br J Cancer 105(11), 1741-49. [article]
  5. Wang F, Wang L, Briggs C, Sicinska E, Gaston SM, Mamon H, Kulke MH, Zamponi R, Loda M, Maher E, Ogino S, Fuchs CS, Li J, Hader C, Makrigiorgos GM. (2007) DNA degradation test predicts success in whole-genome amplification from diverse clinical samples. J Mol Diagn 9(4):441-51. [article]
  6. von Ahlfen S, Missel A, Bendrat K, Schlumpberger M. (2007) Determinants of RNA quality from FFPE samples. PLoS ONE 2(12):e1261. [article]
  7. Doleshal M, Magotra AA, Choudhury B, Cannon BD, Labourier E, Szafranska AE. (2008) Evaluation and validation of total RNA extraction methods for MicroRNA expression analyses in formalin-fixed, paraffin-embedded tissues. J Mol Diagn 10(3):203-11. [article]
  8. LC Sciences internal research.
  9. Li J, Smyth P, Flavin R, Cahill S, Denning K, Aherne S, Guenther SM, O’Leary JJ, Sheils O. (2007) 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 29;7:36. [article]
  10. Hoefig KP, Thorns C, Roehle A, Kaehler C, Wesche KO, Repsilber D, Branke B, Thiere M, Feller AC, Merz H. (2008) Unlocking pathology archives for microRNA-profiling. Anticancer Res 28(1A):119-23. [article]
  11. Laios A, O’Toole S, Flavin R, Martin C, Kelly L, Ring M, Finn SP, Barrett C, Loda M, Gleeson N, D’Arcy T, McGuinness E, Sheils O, Sheppard B, O’ Leary J. (2008) Potential role of miR-9 and miR-223 in recurrent ovarian cancer. Mol Cancer 7:35. [article]
  12. Xi Y, Nakajima G, Gavin E, Morris CG, Kudo K, Hayashi K, Ju J. (2007) Systematic analysis of microRNA expression of RNA extracted from fresh frozen and formalin-fixed paraffin-embedded samples. RNA 13(10):1668-74. [article]
  13. Varnholt H, Drebber U, Schulze F, Wedemeyer I, Schirmacher P, Dienes HP, Odenthal M. (2008) MicroRNA gene expression profile of hepatitis C virus-associated hepatocellular carcinoma. Hepatology 47(4):1223-32. [article]

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Application Note

Archiving histological tissue samples by formalin-fixing and embedding in paraffin blocks has been a routine practice for medical and research institutions for many years and large collections of these samples now exist throughout the world.  Estimates of sample numbers range from 300-400 million in the US alone covering virtually all known diseases. This number may be growing by tens of millions of samples per year.1

What makes these samples of particular interest is the rich patient data and medical history associated with each sample.  Because the samples are so well annotated, they are proving to be a valuable source of information for retrospective clinical studies of human diseases.

Initially, it was unclear if the nucleic acids would remain in sufficient condition to provide robust analytical results due to the adverse effects of formalin fixation.  But studies have shown that although degradation to large RNA can occur, in general, small RNAs are unaffected by the fixation process2.  Indeed there are now a multitude of commercially available kits and methods for extracting microRNA from FFPE prepared tissue samples.3

Furthermore, it has been demonstrated in multiple studies that miRNA is minimally affected by FFPE treatment and microRNA extracted from FFPE samples provides reliable expression levels by direct comparison with frozen tissue samples.4,5,6,7,8 MicroRNA expression data was found to be reliable for FFPE samples stored for up to 30 years.9

Example Data:  FFPE vs Frozen Sample Expression Results

Kits for extraction of microRNA from FFPE prepared tissue samples:

  • RecoverAll Total Nucleic Acid Isolation Kit for FFPE – Ambion
  • miRNeasy FFPE Kit (50) – Qiagen
  • FFPE RNA Purification Kit – Norgen

LC Sciences Customer Publications

  1. Magrelli A, Azzalin G, Salvatore M, Viganotti M, Tosto F, Colombo T, Devito R, Di Masi A, Antoccia A, Lorenzetti S, Maranghi F, Mantovani A, Tanzarella C, Macino G, Taruscio D. (2009) Altered microRNA Expression Patterns in Hepatoblastoma Patients. Transl Oncol 2(3), 157-63. [article]
  2. Zhao Y, Deng C, Wang J, Xiao J, Gatalica Z, Recker RR, Xiao GG. (2010) Let-7 family miRNAs regulate estrogen receptor alpha signaling in estrogen receptor positive breast cancer. Breast Cancer Res Treat 127(1), 69-80. [article]
  3. Zhu Y, Yu X, Fu H, Wang H, Wang P, Zheng X, Wang Y. (2010) MicroRNA-21 is involved in ionizing radiation-promoted liver carcinogenesis. Int J Clin Exp Med 3(3), 211-22. [article]
  4. White NM, Khella HW, Grigull J, Adzovic S, Youssef YM, Honey RJ, Stewart R, Pace KT, Bjarnason GA, Jewett MA, Evans AJ, Gabril M, Yousef GM. (2011) miRNA profiling in metastatic renal cell carcinoma reveals a tumour-suppressor effect for miR-215. Br J Cancer 105(11), 1741-49. [article]
  5. Wang F, Wang L, Briggs C, Sicinska E, Gaston SM, Mamon H, Kulke MH, Zamponi R, Loda M, Maher E, Ogino S, Fuchs CS, Li J, Hader C, Makrigiorgos GM. (2007) DNA degradation test predicts success in whole-genome amplification from diverse clinical samples. J Mol Diagn 9(4):441-51. [article]
  6. von Ahlfen S, Missel A, Bendrat K, Schlumpberger M. (2007) Determinants of RNA quality from FFPE samples. PLoS ONE 2(12):e1261. [article]
  7. Doleshal M, Magotra AA, Choudhury B, Cannon BD, Labourier E, Szafranska AE. (2008) Evaluation and validation of total RNA extraction methods for MicroRNA expression analyses in formalin-fixed, paraffin-embedded tissues. J Mol Diagn 10(3):203-11. [article]
  8. LC Sciences internal research.
  9. Li J, Smyth P, Flavin R, Cahill S, Denning K, Aherne S, Guenther SM, O’Leary JJ, Sheils O. (2007) 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 29;7:36. [article]
  10. Hoefig KP, Thorns C, Roehle A, Kaehler C, Wesche KO, Repsilber D, Branke B, Thiere M, Feller AC, Merz H. (2008) Unlocking pathology archives for microRNA-profiling. Anticancer Res 28(1A):119-23. [article]
  11. Laios A, O’Toole S, Flavin R, Martin C, Kelly L, Ring M, Finn SP, Barrett C, Loda M, Gleeson N, D’Arcy T, McGuinness E, Sheils O, Sheppard B, O’ Leary J. (2008) Potential role of miR-9 and miR-223 in recurrent ovarian cancer. Mol Cancer 7:35. [article]
  12. Xi Y, Nakajima G, Gavin E, Morris CG, Kudo K, Hayashi K, Ju J. (2007) Systematic analysis of microRNA expression of RNA extracted from fresh frozen and formalin-fixed paraffin-embedded samples. RNA 13(10):1668-74. [article]
  13. Varnholt H, Drebber U, Schulze F, Wedemeyer I, Schirmacher P, Dienes HP, Odenthal M. (2008) MicroRNA gene expression profile of hepatitis C virus-associated hepatocellular carcinoma. Hepatology 47(4):1223-32. [article]

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